Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof

ABSTRACT

This invention relates to a reproducible system for the production of stable, genetically transformed maize cells, and to methods of selecting cells that have been transformed. One method of selection disclosed employs the Streptomyces bar gene introduced by microprojectile bombardment into embryogenic maize cells which were grown in suspension cultures, followed by exposure to the herbicide bialaphos. The methods of achieving stables transformation disclosed herein include tissue culture methods and media, methods for the bombardment of recipient cells with the desired transforming DNA, and methods of growing fertile plants from the transformed cells. This invention also relates to the transformed cells and seeds and to the fertile plants grown from the transformed cells and to their pollen.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/112,245, filed Aug.25, 1993, which is a continuation-in-part of U.S. Ser. No. 07/636,089,filed Dec. 28, 1990, now abandoned, which was a continuation-in-part ofU.S. Ser. No. 07/508,045, filed Apr. 11, 1990, now U.S. Pat. No.5,484,956, which was a continuation-in-part of U.S. Ser. No. 07/467,983,filed Jan. 22, 1990, now abandoned. These applications are expresslyincorporated by reference into the application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to reproducible systems for geneticallytransforming monocotyledonous plants such as maize, to methods ofselecting stable genetic transformants from suspensions of transformedcells, and to methods of producing fertile plants from the transformedcells. Exemplary transformation methods include the use ofmicroprojectile bombardment to introduce nucleic acids into cells, andselectable and/or screenable marker systems, for example, genes whichconfer resistance (e.g., antibiotic, herbicide, etc.), or which containan otherwise phenotypically observable or other detectable trait. Inother aspects, the invention relates to the production of stablytransformed and fertile monocot plants, gametes and offspring from thetransgenic plants.

2. Description of the Related Art

Ever since the human species emerged from the hunting-gathering phase ofits existence, and entered an agricultural phase, a major goal of humaningenuity and invention has been to improve crop yield and to alter andimprove the characteristics of plants. In particular, man has sought toalter the characteristics of plants to make them more tasty and/ornutritious, to produce increased crop yield or render plants moreadaptable to specific environments.

Up until recent times, crop and plant improvements depended on selectivebreeding of plants with desirable characteristics. Initial breedingsuccess was probably accidental, resulting from observation of a plantwith desirable characteristics, and use of that plant to propagate thenext generation. However, because such plants had within themheterogenous genetic complements, it was unlikely that progeny identicalto the parent(s) with the desirable traits would emerge. Nonetheless,advances in controlled breeding have resulted from both increasingknowledge of the mechanisms operative in hereditary transmission, and byempirical observations of results of making various parental plantcrosses.

Recent advances in molecular biology have dramatically expanded man'sability to manipulate the germplasm of animals and plants. Genescontrolling specific phenotypes, for example specific polypeptides thatlend antibiotic or herbicide resistance, have been located withincertain germplasm and isolated from it. Even more important has been theability to take the genes which have been isolated from one organism andto introduce them into another organism. This transformation may beaccomplished even where the recipient organism is from a differentphylum, genus or species from that which donated the gene (heterologoustransformation).

Attempts have been made to genetically engineer desired traits intoplant genomes by introduction of exogenous genes using geneticengineering techniques. These techniques have been successfully appliedin some plant systems, principally in dicotyledonous species. The uptakeof new DNA by recipient plant cells has been accomplished by variousmeans, including Agrobacterium infection (Nester et al., 1984),polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985),electroporation of protoplasts (Fromm et al., 1986) and microprojectilebombardment (Klein et al., 1987). Unfortunately, the introduction ofexogenous DNA into monocotyledonous species and subsequent regenerationof transformed plants has proven much more difficult than transformationand regeneration in dicotyledonous plants. Moreover, reports of methodsfor the transformation of monocotyledons such as maize, and subsequentproduction of fertile maize plants, have not been forthcoming.Consequently, success has not been achieved in this area and commercialimplementation of transformation by production of fertile transgenicplants has not been achieved. This failure has been particularlyunfortunate in the case of maize, where there is a particularly greatneed for methods for improving genetic characteristics.

Problems in the development of genetically transformed monocotyledonousspecies have arisen in a variety of general areas. For example, there isgenerally a lack of methods which allow one to introduce nucleic acidsinto cells and yet permit efficient cell culture and eventualregeneration of fertile plants. Only limited successes have been noted.In rice, for example, DNA transfer has only recently been reported usingprotoplast electroporation and subsequent regeneration of transgenicplants (Shimamoto et al., 1989). Furthermore, in maize, transformationusing protoplast electroporation has also been reported (see, e.g.,Fromm et al., 1986).

However, recovery of stably transformed plants has not beenreproducible. A particularly serious failure is that the few transgenicplants produced in the case of maize have not been fertile (Rhodes etal., 1988). While regeneration of fertile corn plants from protoplastshas been reported (Prioli & Sondahl, 1989; Shillito et al., 1989), thesereported methods have been limited to the use of non-transformedprotoplasts. Moreover, regeneration of plants from protoplasts is atechnique which carries its own set of significant drawbacks. Even withvigorous attempts to achieve fertile, transformed maize plants, reportsof success in this regard have not been forthcoming.

A transformation technique that circumvents the need to use protoplastsis microprojectile bombardment. Although transient expression of areporter gene was detected in bombarded tobacco pollen (Twell et al.,1989), stable transformation by microprojectile bombardment of pollenhas not been reported for any plant species. Bombardment of soybeanapical meristems with DNA-coated gold particles resulted in chimericplants containing transgenic sectors. Progeny containing the introducedgene were obtained at a low frequency (McCabe et al., 1988). Bombardmentof shoot meristems of immature maize embryos resulted in sectors oftissue expressing a visible marker, anthocyanin, the synthesis of whichwas triggered by the introduction of a regulatory gene (Tomes, 1990). Ananalysis of cell lineage patterns in maize (McDaniel & Poethig, 1988)suggests that germline transformation of maize by such an approach maybe difficult.

A second major problem in achieving successful monocot transformationhas resulted from the lack of efficient marker gene systems which havebeen employed to identify stably transformed cells. Marker gene systemsare those which allow the selection of, and/or screening for, expressionproducts of DNA. For use as assays for transformed cells, the selectableor screenable products should be those from genetic constructsintroduced into the recipient cells. Hence, such marker genes can beused to identify stable transformants.

Of the more commonly used marker gene systems are gene systems whichconfer resistance to aminoglycosides such as kanamycin. While kanamycinresistance has been used successfully in both rice (Yang et al., 1988)and corn protoplast systems (Rhodes et al., 1988), it remains a verydifficult selective agent to use in monocots due to high endogenousresistance (Hauptmann, et al., 1988). Many monocot species, maize, inparticular, possess high endogenous levels of resistance toaminoglycosides. Consequently, this class of compounds cannot be usedreproducibly to distinguish transformed from non-transformed tissue. Newmethods for reproducible selection of or screening for transformed plantcells are therefore needed.

Accordingly, it is clear that improved methods and/or approaches to thegenetic transformation of monocotyledonous species would represent agreat advance in the art. Furthermore, it would be of particularsignificance to provide novel approaches to monocot transformation, suchas transformation of maize cells, which would allow for the productionof stably transformed, fertile corn plants and progeny into whichdesired exogenous genes have been introduced. Furthermore, theidentification of marker gene systems applicable to monocot systems suchas maize would provide a useful means for applying such techniquesgenerally. Thus, the development of these and other techniques for thepreparation of stable genetically transformed monocots such as maizecould potentially revolutionize approaches to monocot breeding.

SUMMARY OF THE INVENTION

The present invention addresses one or more of the foregoing or othershortcomings in the prior art by providing compositions and methods forthe preparation of stably transformed, monocotyledonous cells and thesubsequent regeneration of fertile, transgenic plants and progeny,particularly maize (Zea mays). The invention particularly providestechniques for the preparation of transgenic, fertile monocots, such asmaize, which have been stably transformed through the introduction ofdiscrete DNA sequences into the plant genome.

Thus, a preferred embodiment of the present invention is a fertiletransgenic Zea mays plant of the R0 generation, the genome of which hasbeen stably augmented by chromosomally integrated DNA, which encodesonly one or more preselected proteins, RNA-transcripts or mixturesthereof, wherein said DNA is expressed so that the transgenic plantexhibits one or more phenotypic characteristics that render itidentifiable over the corresponding untransformed Zea mays plant whichdoes not comprise said DNA, and wherein the DNA is transmitted through anormal sexual cycle of the transgenic Zea mays plant to the R1generation of plants.

For example, the DNA can be expressed so that the transgenic plant isresistant to an amount of an agent such as a herbicide, which would betoxic to a Zea mays plant which does not comprise said gene. Thetransgenic Zea mays plants of the present invention are also distinctfrom genetically altered or "improved" Zea mays plants which have beenproduced by prior art methods. For example, conventional maize breedingtechniques randomly introduce large amounts of genetic material fromboth parents into the progeny plants. In contrast, the genome of theregenerant or R0 maize plant of the present invention has been augmentedby "foreign" or "exogenous" plant or non-plant DNA which encodes one ormore proteins, RNA transcripts or mixtures thereof that can bepreselected by the art worker. Thus, the number sizes and/or thefunctional characteristics, if not the precise and preferably, thestructures of both the introduced DNA and the products encoded therebycan be controlled, delimited and/or defined.

The Zea mays plants of the present invention are also necessarilydistinct from Zea mays plants which comprise one or more "native" geneswhich have been altered or deleted by random in vivo mutation, as bychemical mutagens or by radiation.

The invention thus relates generally to methods for the production oftransgenic plants. As used herein, the term "transgenic plants" isintended to refer to plants, the genome of which has been augmented byat least one incorporated DNA sequence. Such DNA sequences include butare not limited to genes which are perhaps not normally present, DNAsequences not normally transcribed into RNA or translated into a protein("expressed"), or any other genes or DNA sequences which one desires tointroduce into the non-transformed plant, such as genes which maynormally be present in the non-transformed plant but which one desiresto either genetically engineer or to alter the expression thereof. It iscontemplated that in some instances the genome of transgenic plants ofthe present invention will have been augmented through the stableintroduction of the transgene. However, in other instances, theintroduced gene will replace an endogenous sequence.

Exemplary genes which may be introduced include, for example, DNAsequences or genes from another species, or even genes or sequenceswhich originate with or are present in the same species, but areincorporated into recipient cells by genetic engineering methods ratherthan classical reproduction or breeding techniques. However, the term"exogenous", is also intended to refer to DNA sequences or genes whichare not normally present in the cell being transformed, or perhaps aresimply not present in the form, structure, etc., as found in thetransforming DNA segment or gene, or genes which are normally presentyet which one desires to alter functionally, e.g., to haveoverexpressed. Thus, the term "exogenous" gene or DNA refers to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell."Introduced", or "augmented" in this context, is known in the art tomean introduced or augmented by the hand of man.

The most preferred monocots for use in the present invention will becereals such as maize (Zea mays). The present invention is exemplifiedthrough the use of A188×B73 cell lines, cell lines developed from othergenotypes and immature embryos. However, it is to be understood that theinvention is in no way limited to a particular genotype or cell line. Todate, a variety of different Zea mays lines and germplasms have beentested for their ability to be successfully employed in the preparationof fertile, transgenic corn. The status of these studies is set forth insome detail hereinbelow. Generally speaking, these studies havedemonstrated that 24 out of 36 maize cultures were transformable. Ofthose cell lines tested, 11 out of 20 have produced fertile plants.

One exemplary embodiment for generating a stably transformed monocotincludes culturing recipient corn cells in suspension cultures usingembryogenic cells in Type II callus, selecting for small (10-30 μ)isodiametric, cytoplasmically dense cells, introducing a desired DNAsegment into these cells, growing the transformed cells in or on culturemedium containing hormones, subculturing into a progression of media tofacilitate development of shoots and roots, and finally, hardening thetransgenic plant and readying it metabolically for growth in soil.

The present invention is suitable for use in transforming any maizevariety. While not all cell lines developed out of a particular varietyor cross will necessarily show the same degree of stabletransformability, it has been the inventors' finding that a reasonablepercentage of cell lines developed from essentially every genotypetested to date can be developed into fertile, transgenic plants. Thus,where one desires to prepare transformants in a particular cross orvariety, it will generally be desirable to develop several cell linesfrom the particular cross or variety (e.g., 8 to 10), and subject all ofthe lines so developed to the transformation protocols hereof.

Another exemplary embodiment for generating a stably transformed monocotincludes introducing a desired DNA segment into cells of organizedtissues such as immature embryos, growing the embryos on a culturemedium , subculturing into a progression of media to facilitatedevelopment of shoots and roots, and finally, hardening the transgenicplant and readying it metabolically for growth in soil. In thisembodiment the invention is capable of transforming any variety ofmaize. Through the use of the present invention it is possible tosimultaneously deliver DNA segments to a large number of embryos. It hasbeen the inventor's finding that a percentage of the embryos that arecontacted by exogenous DNA will develop into fertile transgenic plants,similar to delivering DNA to a large population of cultured cells. Thepresent invention is exemplified through the use of immature embryosfrom the genotypes H99 and Hi-II, but is in no way limited to thesegenotypes. To date only experiments with these genotypes have progressedto the point where one would reasonably expect to recover transformants.Generally speaking one would expect to be able to recover fertiletransgenic plants from any variety of maize.

Moreover, the ability to provide even a single fertile, transgenic cornline would be generally sufficient to allow the introduction of thetransgenic component (e.g., recombinant DNA) of that line into a secondcorn line of choice. This is because by providing fertile, transgenicoffspring, the practice of the invention allows one to subsequently,through a series of breeding manipulations, move a selected gene fromone corn line into an entirely different corn line. For example, studieshave been conducted wherein the gene for resistance to the herbicideBasta®, bar, has been moved from two transformants derived from cellline SC716 and one transformant derived from cell line SC82 into 18elite inbred lines by backcrossing. It is possible with these inbreds toproduce a large number of hybrids. Eleven such hybrids have been madeand are in field tests.

I. Recipient Cells

Practicing the present invention includes the generation and use ofrecipient cells. As used herein, the term "recipient cells" refers tomonocot cells that are receptive to transformation and subsequentregeneration into stably transformed, fertile monocot plants.

A. Sources of Cells

Recipient cell targets include, but are not limited to, meristem cells,Type I, Type II, and Type III callus, immature embryos and gametic cellssuch as microspores pollen, sperm and egg cells. Type I, Type II, andType III callus may be initiated from tissue sources including, but notlimited to, immature embryos, seedling apical meristems, microspores andthe such. Those cells which are capable of proliferating as callus arealso recipient cells for genetic transformation. The present inventionprovides techniques for transforming immature embryos followed byinitiation of callus and subsequent regeneration of fertile transgenicplants. Direct transformation of immature embryos obviates the need forlong term development of recipient cell cultures. Pollen, as well as itsprecursor cells, microspores, may be capable of functioning as recipientcells for genetic transformation, or as vectors to carry foreign DNA forincorporation during fertilization. Direct pollen transformation wouldobviate the need for cell culture. Meristematic cells (i.e., plant cellscapable of continual cell division and characterized by anundifferentiated cytological appearance, normally found at growingpoints or tissues in plants such as root tips, stem apices, lateralbuds, etc.) may represent another type of recipient plant cell. Becauseof their undifferentiated growth and capacity for organ differentiationand totipotency, a single transformed meristematic cell could berecovered as a whole transformed plant. In fact, it is proposed thatembryogenic suspension cultures may be an in vitro meristematic cellsystem, retaining an ability for continued cell division in anundifferentiated state, controlled by the media environment.

In certain embodiments, cultured plant cells that can serve as recipientcells for transforming with desired DNA segments include corn cells, andmore specifically, cells from Zea mays L. Somatic cells are of varioustypes. Embryogenic cells are one example of somatic cells which may beinduced to regenerate a plant through embryo formation. Non-embryogeniccells are those which will typically not respond in such a fashion. Anexample of non-embryogenic cells are certain Black Mexican Sweet (BMS)corn cells. These cells have been transformed by microprojectilebombardment using the neo gene followed by selection with theaminoglycoside, kanamycin (Klein et al., 1989). However, this BMSculture was not found to be regenerable.

The development of embryogenic maize calli and suspension culturesuseful in the context of the present invention, e.g., as recipient cellsfor transformation, has been described in U.S. Ser. No. 06/877,033,filed Jun. 7, 1986, incorporated herein by reference.

The present invention also provides certain techniques that may enrichrecipient cells within a cell population. For example, Type II callusdevelopment, followed by manual selection and culture of friable,embryogenic tissue, generally results in an enrichment of recipientcells for use in, e.g., micro-projectile transformation. Suspensionculturing, particularly using the media disclosed herein, may alsoimprove the ratio of recipient to non-recipient cells in any givenpopulation. Manual selection techniques which employed to selectrecipient cells may include, e.g., assessing cell morphology anddifferentiation, or may use various physical or biological means.Cryopreservation is also contemplated as a possible method of selectingfor recipient cells.

Manual selection of recipient cells, e.g., by selecting embryogeniccells from the surface of a Type II callus, is one means employed by theinventors in an attempt to enrich for recipient cells prior to culturing(whether cultured on solid media or in suspension). The preferred cellsmay be those located at the surface of a cell cluster, and may furtherbe identifiable by their lack of differentiation, their size and densecytoplasm. The preferred cells will generally be those cells which areless differentiated, or not yet committed to differentiation. Thus, onemay wish to identify and select those cells which are cytoplasmicallydense, relatively unvacuolated with a high nucleus to cytoplasm ratio(e.g., determined by cytological observations), small in size (e.g.,10-20 μm), and capable of sustained divisions and somatic proembryoformation.

It is proposed that other means for identifying such cells may also beemployed. For example, through the use of dyes, such as Evan's blue,which are excluded by cells with relatively non-permeable membranes,such as embryogenic cells, and taken up by relatively differentiatedcells such as root-like cells and snake cells (so-called due to theirsnake-like appearance).

Other possible means of identifying recipient cells include the use ofisozyme markers of embryogenic cells, such as glutamate dehydrogenase,which can be detected by cytochemical stains (Fransz et al., 1989).However, it is cautioned that the use of isozyme markers such asglutamate dehydrogenase may lead to some degree of false positives fromnon-embryogenic cells such as rooty cells which nonetheless have arelatively high metabolic activity.

B. Media

In certain embodiments, recipient cells are selected following growth inculture. Where employed, cultured cells will preferably be grown eitheron solid supports or in the form of liquid suspensions. In eitherinstance, nutrients may be provided to the cells in the form of media,and environmental conditions controlled. There are many types of tissueculture media comprised of amino acids, salts, sugars, growth regulatorsand vitamins. Most of the media employed in the practice of theinvention will have some similar components (see, e.g., Table 1 hereinbelow), the media differ in the composition and proportions of theiringredients depending on the particular application envisioned. Forexample, various cell types usually grow in more than one type of media,but will exhibit different growth rates and different morphologies,depending on the growth media. In some media, cells survive but do notdivide.

Various types of media suitable for culture of plant cells have beenpreviously described. Examples of these media include, but are notlimited to, the N6 medium described by Chu et al. (1975) and MS media(Murashige & Skoog, 1962). The inventors have discovered that media suchas MS which have a high ammonia/nitrate ratio are counterproductive tothe generation of recipient cells in that they promote loss ofmorphogenic capacity. N6 media, on the other hand, has a somewhat lowerammonia/nitrate ratio, and is contemplated to promote the generation ofrecipient cells by maintaining cells in a proembryonic state capable ofsustained divisions.

C. Cell Cultures

1. Initiation

In the practice of the invention it is sometimes, but not always,necessary to develop cultures which contain recipient cells. Suitablecultures can be initiated from a number of whole plant tissue explantsincluding, but not limited to, immature embryos, leaf bases, immaturetassels, anthers, microspores, and other tissues containing cellscapable of in vitro proliferation and regeneration of fertile plants. Inone exemplary embodiment, recipient cell cultures are initiated fromimmature embryos of Zea mays L. by growing excised immature embryos on asolid culture medium containing growth regulators including, but notlimited to, dicamba., 2,4-D, NAA, and IAA. In some instances it will bepreferred to add silver nitrate to culture medium for callus initiationas this compound has been reported to enhance culture initiation (Vainet al., 1989). Embryos will produce callus that varies greatly inmorphology including from highly unorganized cultures containing veryearly embryogenic structures (such as, but not limited to, type IIcultures in maize), to highly organized cultures containing large lateembryogenic structures (such as, but not limited to, type I cultures inmaize). This variation in culture morphology may be related to genotype,culture medium composition, size of the initial embryos and otherfactors. Each of these types of culture morphologies is a source ofrecipient cells.

The development of suspension cultures capable of plant regeneration maybe used in the context of the present invention. Suspension cultures maybe initiated by transferring callus tissue to liquid culture mediumcontaining growth regulators. Addition of coconut water or othersubstances to suspension culture medium may enhance growth and culturemorphology, but the utility of suspension cultures is not limited tothose containing these compounds. In some embodiments of this invention,the use of suspension cultures will be preferred as these cultures growmore rapidly and are more easily manipulated than callus cells growingon solid culture medium.

When immature embryos or other tissues directly removed from a wholeplant are used as the target tissue for DNA delivery, it will only benecessary to initiate cultures of cells insofar as is necessary foridentification and isolation of transformants. In an illustrativeembodiment, DNA is introduced by particle bombardment into immatureembryos following their excision from the plant. Embryos are transferredto a culture medium that will support proliferation of tissues and allowfor selection of transformed sectors, 0-14 days following DNA delivery.In this embodiment of the invention it is not necessary to establishstable callus cultures capable of long term maintenance and plantregeneration.

2. Maintenance

The method of maintenance of cell cultures may contribute to theirutility as sources of recipient cells for transformation. Manualselection of cells for transfer to fresh culture medium, frequency oftransfer to fresh culture medium, composition of culture medium, andenvironment factors including, but not limited to, light quality andquantity and temperature are all important factors in maintaining callusand/or suspension cultures that are useful as sources of recipientcells. It is contemplated that alternating callus between differentculture conditions may be beneficial in enriching for recipient cellswithin a culture. For example, it is proposed that cells may be culturedin suspension culture, but transferred to solid medium at regularintervals. After a period of growth on solid medium cells can bemanually selected for return to liquid culture medium. It is proposedthat by repeating this sequence of transfers to fresh culture medium itis possible to enrich for recipient cells. It is also contemplated thatpassing cell cultures through a 1.9 mm sieve is useful in maintainingthe friability of a callus or suspension culture and may be beneficialis enriching for transformable cells.

3. Cryopreservation

Additionally, the inventors propose that cryopreservation may effect thedevelopment of, or perhaps select for, recipient cells. Cryopreservationselection may operate due to a selection against highly vacuolated,non-embryogenic cells, which may be selectively killed duringcryopreservation. The inventors propose that there is a temporal windowin which cultured cells retain their regenerative ability, thus, it isbelieved that they must be preserved at or before that temporal periodif they are to be used for future transformation and regeneration.

For use in transformation, suspension or callus culture cells may becryopreserved and stored for periods of time, thawed, then used asrecipient cells for transformation. An illustrative embodiment ofcryopreservation methods comprises the steps of slowly addingcryoprotectants to suspension cultures to give a final concentration of10% dimethyl sulfoxide, 10% polyethylene glycol (6000 MW), 0.23 Mproline and 0.23 M glucose. The mixture is then cooled to -35° C. at0.5° C. per minute. After an isothermal period of 45 minutes, samplesare placed in liquid N₂ (modification of methods of Withers and King(1979); and Finkle et al. (1985)). To reinitiate suspension culturesfrom cryopreserved material, cells may be thawed rapidly and pipettedonto feeder plates similar to those described by Rhodes et al. (Vaeck etal., 1987).

II. DNA Sequences

Virtually any DNA composition may be used for delivery to recipientmonocotyledonous cells to ultimately produce fertile transgenic plantsin accordance with the present invention. For example, DNA segments inthe form of vectors and plasmids, or linear DNA fragments, in someinstances containing only the DNA element to be expressed in the plant,and the like, may be employed.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) "shuttle" vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in maize cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs et al., 1990) that transpositionof these elements within the maize genome requires DNA replication. Itis also contemplated that transposable elements would be useful forintroducing DNA fragments lacking elements necessary for selection andmaintenance of the plasmid vector in bacteria, e.g., antibioticresistance genes and origins of DNA replication.

It is also proposed that use of a transposable element such as Ac, Ds,or Mu would actively promote integration of the desired DNA and henceincrease the frequency of stably transformed cells.

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes) and DNAsegments for use in transforming such cells will, of course, generallycomprise the cDNA, gene or genes which one desires to introduce into thecells. These DNA constructs can further include structures such aspromoters, enhancers, polylinkers, or even regulatory genes as desired.The DNA segment or gene chosen for cellular introduction will oftenencode a protein which will be expressed in the resultant recombinantcells, such as will result in a screenable or selectable trait and/orwhich will impart an improved phenotype to the regenerated plant.However, this may not always be the case, and the present invention alsoencompasses transgenic plants incorporating non-expressed transgenes.

DNA useful for introduction into Zea mays cells includes that which hasbeen derived or isolated from any source, that may be subsequentlycharacterized as to structure, size and/or function, chemically altered,and later introduced into Zea mays. An example of DNA "derived" from asource, would be a DNA sequence that is identified as a useful fragmentwithin a given organism, and which is then chemically synthesized inessentially pure form. An example of such DNA "isolated" from a sourcewould be a useful DNA sequence that is excised or removed from saidsource by chemical means, e.g., by the use of restriction endonucleases,so that it can be further manipulated, e.g., amplified, for use in theinvention, by the methodology of genetic engineering. Such DNA iscommonly referred to as "recombinant DNA."

Therefore useful DNA includes completely synthetic DNA, semi-syntheticDNA, DNA isolated from biological sources, and DNA derived fromintroduced RNA. Generally, the introduced DNA is not originally residentin the Zea mays genotype which is the recipient of the DNA, but it iswithin the scope of the invention to isolate a gene from a given Zeamays genotype, and to subsequently introduce multiple copies of the geneinto the same genotype, e.g., to enhance production of a given geneproduct such as a storage protein.

The introduced DNA includes but is not limited to, DNA from plant genes,and non-plant genes such as those from bacteria, yeasts, animals orviruses; modified genes, portions of genes, chimeric genes, includinggenes from the same or different Zea mays genotype.

The introduced DNA used for transformation herein may be circular orlinear, double-stranded or single-stranded. Generally, the DNA is in theform of chimeric DNA, such as plasmid DNA, that can also contain codingregions flanked by regulatory sequences which promote the expression ofthe recombinant DNA present in the resultant corn plant. For example,the DNA may itself comprise or consist of a promoter that is active inZea mays, or may utilize a promoter already present in the Zea maysgenotype that is the transformation target. Generally, the introducedDNA will be relatively small, i.e., less than about 30 kb to minimizeany susceptibility to physical, chemical, or enzymatic degradation whichis known to increase as the size of the DNA increases. As noted above,the number of proteins, RNA transcripts or mixtures thereof which isintroduced into the Zea mays genome is preferably preselected anddefined, e.g., from one to about 5-10 such products of the introducedDNA may be formed.

A. Regulatory Elements

The construction of vectors which may be employed in conjunction withthe present invention will be known to those of skill of the art inlight of the present disclosure (see e.g., Sambrook et al., 1989; Gelvinet al., 1990). Preferred constructs will generally include a plantpromoter such as the CaMV 35S promoter (Odell et al., 1985), or otherssuch as CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh(Walker et al., 1987), sucrose synthase (Yang & Russell, 1990),α-tubulin, actin (Wang et al., 1992), cab (Sullivan et al., 1989),PEPCase (Hudspeth & Grula, 1989) or those associated with the R genecomplex (Chandler et al., 1989). Tissue specific promoters such as rootcell promoters (Conkling et al., 1990) and tissue specific enhancers(Fromm et al., 1989) are also contemplaetd to be particularly useful, asare inducible promoters such as ABA- and turgor-inducible promters.

Constructs will also include the gene of interest along with a 3' endDNA sequence that acts as a signal to terminate transcription and allowfor the poly-adenylation of the resultant mRNA. The most preferred 3'elements are contemplated to be those from the nopaline synthase gene ofAgrobacterium tumefasciens (Bevan et al., 1983), the terminator for theT7 transcript from the octopine synthase gene of Agrobacteriumtumefasciens, and the 3' end of the protease inhibitor I or II genesfrom potato or tomato. Regulatory elements such as Adh intron 1 (Calliset al., 1987), sucrose synthase intron (Vasil et al., 1989) or TMV omegaelement (Gallie, et al., 1989), may further be included where desired.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants, and in maize in particular, will be most preferred.

It is contemplated that vectors for use in accordance with the presentinvention may be constructed to include the ocs enhancer element. Thiselement was first identified as a 16 bp palindromic enhancer from theoctopine synthase (ocs) gene of agrobacterium (Ellis et al., 1987), andis present in at least 10 other promoters (Bouchez et al., 1989). It isproposed that the use of an enhancer element, such as the ocs elementand particularly multiple copies of the element, will act to increasethe level of transcription from adjacent promoters when applied in thecontext of monocot transformation.

Ultimately, the most desirable DNA segments for introduction into amonocot genome may be homologous genes or gene families which encode adesired trait (e.g., increased yield per acre) and which are introducedunder the control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue specific (e.g., root-, collar/sheath-, whorl-,stalk-, earshank-, kernel- or leaf-specific) promoters or controlelements. Indeed, it is envisioned that a particular use of the presentinvention will be the targeting of a gene in a tissue-specific manner.For example, insect resistant genes may be expressed specifically in thewhorl and collar/sheath tissues which are targets for the first andsecond broods, respectively, of ECB. Likewise, genes encoding proteinswith particular activity against rootworm may be targeted directly toroot tissues.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(-90 to +8) 35S promoter which directs enhanced expression in roots, anα-tubulin gene that directs expression in roots and promoters derivedfrom zein storage protein genes which direct expression in endosperm. Itis particularly contemplated that one may advantageously use the 16 bpocs enhancer element from the octopine synthase (ocs) gene (Ellis etal., 1987; Bonchez et al., 1989), especially when present in multiplecopies, to achieve enhanced expression in roots.

It is also contemplated that tissue specific expression may befunctionally accomplished by introducing a constituitively expressedgene (all tissues) in combination with an antisense gene that isexpressed only in those tissues where the gene product is not desired.For example, a gene coding for the crystal toxin protein from B.thuringiensis (Bt) may be introduced such that it is expressed in alltissues using the 35S promoter from Cauliflower Mosaic Virus. Expressionof an antisense transcript of the Bt gene in a maize kernel, using forexample a zein promoter, would prevent accumulation of the Bt protein inseed. Hence the protein encoded by the introduced gene would be presentin all tissues except the kernel.

Alternatively, one may wish to obtain novel tissue-specific promotersequences for use in accordance with the present invention. To achievethis, one may first isolate cDNA clones from the tissue concerned andidentify those clones which are expressed specifically in that tissue,for example, using Northern blotting. Ideally, one would like toidentify a gene that is not present in a high copy number, but whichgene product is relatively abundant in specific tissues. The promoterand control elements of corresponding genomic clones may then belocalized using the techniques of molecular biology known to those ofskill in the art.

It is contemplated that expression of some genes in transgenic plantswill be desired only under specified conditions. For example, it isproposed that expression of certain genes that confer resistance toenvironmental stress factors such as drought will be desired only underactual stress conditions. It is contemplated that expression of suchgenes throughout a plants development may have detrimental effects. Itis known that a large number of genes exist that respond to theenvironment. For example, expression of some genes such as rbcS,encoding the small subunit of ribulose bisphosphate carboxylase, isregulated by light as mediated through phytochrome. Other genes areinduced by secondary stimuli. For example, synthesis of abscisic acid(ABA) is induced by certain environmental factors, including but notlimited to water stress. A number of genes have been shown to be inducedby ABA (Skriver and Mundy, 1990). It is also anticipated that expressionof genes conferring resistance to insect predation would be desired onlyunder conditions of actual insect infestation. Therefore, for somedesired traits inducible expression of genes in transgenic plants willbe desired.

It is proposed that in some embodiments of the present inventionexpression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcS transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

It is also contemplated that it may be useful to target DNA itselfwithin a cell. For example, it may be useful to target introduced DNA tothe nucleus as this may increase the frequency of transformation. Withinthe nucleus itself it would be useful to target a gene in order toacheive site specific integration. For example, it would be useful tohave an gene introduced through transformation replace an existing genein the cell.

B. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. "Marker genes" are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can `select` for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by `screening` (e.g., the R-locus trait). Of course, manyexamples of suitable marker genes are known to the art and can beemployed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a "secretable marker" whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., α-amylase, β-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). The use of themaize HPRG (Steifel et al., 1990) which is preferred as this molecule iswell characterized in terms of molecular biology, expression and proteinstructure. However, any one of a variety of extensins and/orglycine-rich wall proteins (Keller et al., 1989) could be modified bythe addition of an antigenic site to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of the maize genomic clone encoding the wall protein HPRG, modifiedto include the unique 15 residue epitope M A T V P E L N C E M P P S D(seq id no:1) from the pro-region of murine interleukin-1-β (IL-1-β).However, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen:antibody combinations known to those of skill in the art. Theunique extracellular epitope, whether derived from IL-1-β or any otherprotein or epitopic substance, can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure are exemplified in detail through theuse of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth hereinbelow.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed monocot.

1. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, etc.; a bar gene which codes for bialaphos resistance;a mutant aroA gene which encodes an altered EPSP synthase protein(Hinchee et al., 1988) thus conferring glyphosate resistance; anitrilase gene such as bxn from Klebsiella ozaenae which confersresistance to bromoxynil (Stalker et al., 1988); a mutant acetolactatesynthase gene (ALS) which confers resistance to imidazolinone,sulfonylurea or other ALS inhibiting chemicals (European PatentApplication 154,204, 1985); a methotrexate resistant DHFR gene (Thilletet al., 1988), or a dalapon dehalogenase gene that confers resistance tothe herbicide dalapon; or a mutated anthranilate synthase gene thatconfers resistance to 5-methyl tryptophan. Where a mutant EPSP synthasegene is employed, additional benefit may be realized through theincorporation of a suitable chloroplast transit peptide, CTP (EuropeanPatent Application 0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success of the inventors in using this selective systemin conjunction with monocots was particularly surprising because of themajor difficulties which have been reported in transformation of cereals(Potrykus, 1989).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, the inventors have discovered that a particularlyuseful gene for this purpose is the bar or pat genes obtainable fromspecies of Streptomyces (e.g., ATCC No. 21,705). The cloning of the bargene has been described (Murakami et al., 1986; Thompson et al., 1987)as has the use of the bar gene in the context of plants other thanmonocots (De Block et al., 1987; De Block et al., 1989).

2. Screenable Markers

Screenable markers that may be employed include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe, 1978),which encodes an enzyme for which various chromogenic substrates areknown (e.g., PADAC, a chromogenic cephalosporin); a xylE gene (Zukowskyet al., 1983) which encodes a catechol dioxygenase that can convertchromogenic catechols; an α-amylase gene (Ikuta et al., 1990); atyrosinase gene (Katz et al., 1983) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily-detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., 1986), which allows forbioluminescence detection; or even an aequorin gene (Prasher et al.,1985) which may be employed in calcium-sensitive bioluminescencedetection.

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. Maize strains can have one, or as many asfour, R alleles which combine to regulate pigmentation in adevelopmental and tissue specific manner. The present inventors haveapplied a gene from the R gene complex to maize transformation, becausethe expression of this gene in transformed cells does not harm thecells. Thus, an R gene introduced into such cells will cause theexpression of a red pigment and, if stably incorporated, can be visuallyscored as a red sector. If a maize line is carries dominant allelles forgenes encoding the enzymatic intermediates in the anthocyaninbiosynthetic pathway (C2, A1, A2, Bz1 and Bz2), but carries a recessiveallele at the R locus, transformation of any cell from that line with Rwill result in red pigment formation. Exemplary lines include Wisconsin22 which contains the rg-Stadler allele and TR112, a K55 derivativewhich is r-g, b, P1. Alternatively any genotype of maize can be utilizedif the C1 and R alleles are introduced together.

The inventors further propose that R gene regulatory regions may beemployed in chimeric constructs in order to provide mechanisms forcontrolling the expression of chimeric genes. More diversity ofphenotypic expression is known at the R locus than at any other locus(Coe et al., 1988). It is contemplated that regulatory regions obtainedfrom regions 5' to the structural R gene would be valuable in directingthe expression of genes for, e.g., insect resistance, herbicidetolerance or other protein coding regions. For the purposes of thepresent invention, it is believed that any of the various R gene familymembers may be successfully employed (e.g., P, S, Lc, etc.). However,the most preferred will generally be Sn (particularly Sn:bol3). Sn is adominant member of the R gene complex and is functionally similar to theR and B loci in that Sn controls the tissue specific deposition ofanthocyanin pigments in certain seedling and plant cells, therefore, itsphenotype is similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

C. Transgenes for Corn Modification

A particularly important advance of the present invention is that itprovides methods and compositions for the transformation of plant cellswith genes in addition to, or other than, marker genes. Such transgeneswill often be genes that direct the expression of a particular proteinor polypeptide product, but they may also be non-expressible DNAsegments, e.g., transposons such as Ds that do no direct their owntransposition. As used herein, an "expressible gene" is any gene that iscapable of being transcribed into RNA (e.g., mRNA, antisense RNA, etc.)or translated into a protein, expressed as a trait of interest, or thelike, etc., and is not limited to selectable, screenable ornon-selectable marker genes. The invention also contemplates that, whereboth an expressible gene that is not necessarily a marker gene isemployed in combination with a marker gene, one may employ the separategenes on either the same or different DNA segments for transformation.In the latter case, the different vectors are delivered concurrently torecipient cells to maximize cotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding herbicide resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phoshinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes. These genes are particularly contemplated for use in monocottransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

2. Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into monocotyledonous plants suchas maize. Potential insect resistance genes which can be introducedinclude Bacillus thuringiensis crystal toxin genes or Bt genes (Watrudet al., 1985). Bt genes may provide resistance to lepidopteran orcoleopteran pests such as European Corn Borer (ECB). Preferred Bt toxingenes for use in such embodiments include the CryIA(b) and CryIA(c)genes. Endotoxin genes from other species of B. thuringiensis whichaffect insect growth or development may also be employed in this regard.

The poor expression of procaryotic Bt toxin genes in plants is awell-documented phenomenon, and the use of different promoters, fusionproteins, and leader sequences has not led to significant increases inBt protein expression (Vaeck et al., 1989; Barton et al., 1987). It istherefore contemplated that the most advantageous Bt genes for use inthe transformation protocols disclosed herein will be those in which thecoding sequence has been modified to effect increased expression inplants, and more particularly, those in which maize preferred codonshave been used. Examples of such modified Bt toxin genes include thevariant Bt CryIA(b) gene termed IAb6 (Perlak et al., 1991) and thesynthetic CryIA(c) genes termed 1800a and 1800b.

Protease inhibitors may also provide insect resistance (Johnson et al.,1989), and will thus have utility in maize transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered by the present inventors to produce synergisticinsecticidal activity. Other genes which encode inhibitors of theinsects' digestive system, or those that encode enzymes or co-factorsthat facilitate the production of inhibitors, may also be useful. Thisgroup may be exemplified by oryzacystatin and amylase inhibitors such asthose from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla & Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic maize expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccuring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgeneicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming maize to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn et al., 1981). The introduction of genes that can regulatethe production of maysin, and genes involved in the production ofdhurrin in sorghum, is also contemplated to be of use in facilitatingresistance to earworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Avermectin and Abamectin., Campbell, W. C.,Ed., 1989; Ikeda et al., 1987) which may prove particularly useful as acorn rootworm deterrent; ribosome inactivating protein genes; and evengenes that regulate plant structures. Transgenic maize includinganti-insect antibody genes and genes that code for enzymes that cancovert a non-toxic insecticide (pro-insecticide) applied to the outsideof the plant into an insecticide inside the plant are also contemplated.

3. Environment or Stress Resistance

Improvement of corn's ability to tolerate various environmental stressessuch as, but not limited to, drought, excess moisture, chilling,freezing, high temperature, salt, and oxidative stress, can also beeffected through expression of novel genes. It is proposed that benefitsmay be realized in terms of increased resistance to freezingtemperatures through the introduction of an "antifreeze" protein such asthat of the Winter Flounder (Cutler et al., 1989) or synthetic genederivatives thereof. Improved chilling tolerance may also be conferredthrough increased expression of glycerol-3-phosphate acetyltransferasein chloroplasts (Murata et al., 1992; Wolter et al., 1992). Resistanceto oxidative stress (often exacerbated by conditions such as chillingtemperatures in combination with high light intensities) can beconferred by expression of superoxide dismutase (Gupta et al., 1993),and may be improved by glutathione reductase (Bowler et al., 1992). Suchstrategies may allow for tolerance to freezing in newly emerged fieldsas well as extending later maturity higher yielding varieties to earlierrelative maturity zones.

It is contemplated that the expression of novel genes that favorablyeffect plant water content, total water potential, osmotic potential,and turgor will enhance the ability of the plant to tolerate drought. Asused herein, the terms "drought resistance" and "drought tolerance" areused to refer to a plants increased resistance or tolerance to stressinduced by a reduction in water availability, as compared to normalcircumstances, and the ability of the plant to function and survive inlower-water environments. In this aspect of the invention it isproposed, for example, that the expression of genes encoding for thebiosynthesis of osmotically-active solutes may impart protection againstdrought. Within this class are genes encoding for mannitol dehydrogenase(Lee and Saier, 1982) and trehalose-6-phosphate synthase (Kaasen et al.,1992). Through the subsequent action of native phosphatases in the cellor by the introduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al., 1992,1993).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis et al., 1989), and thereforeexpression of genes encoding for the biosynthesis of these compoundsmight confer drought resistance in a manner similar to or complimentaryto mannitol. Other examples of naturally occurring metabolites that areosmotically active and/or provide some direct protective effect duringdrought and/or desiccation include fructose, erythritol (Coxson et al.,1992), sorbitol, dulcitol (Karsten et al., 1992), glucosylglycerol (Reedet al., 1984; Erdmann et al., 1992), sucrose, stachyose (Koster andLeopold, 1988; Blackman et al., 1992), raffinose (Bernal-Lugo andLeopold, 1992), proline (Rensburg et al., 1993) and glycinebetaine(Wyn-Jones and Storey, 1982), ononitol and pinitol (Vernon and Bohnert,1992). Continued canopy growth and increased reproductive fitness duringtimes of stress will be augmented by introduction and expression ofgenes such as those controlling the osmotically active compoundsdiscussed above and other such compounds, as represented in oneexemplary embodiment by the enzyme myoinositol O-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of LEAs have been demonstrated in maturing(i.e. desiccating) seeds. Within these 3 types of LEA proteins, theType-II (dehydrin-type) have generally been implicated in drought and/ordesiccation tolerance in vegetative plant parts (i.e. Mundy and Chua,1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). Expression of structural genes from all three LEA groups maytherefore confer drought tolerance. Other types of proteins inducedduring water stress include thiol proteases, aldolases and transmembranetransporters (Guerrero et al., 1990), which may confer variousprotective and/or repair-type functions during drought stress. It isalso contemplated that genes that effect lipid biosynthesis and hencemembrane composition might also be useful in conferring droughtresistance on the plant.

Many of these genes for improving drought resistance have complementarymodes of action. Thus, it is envisaged that combinations of these genesmight have additive and/or synergistic effects in improving droughtresistance in corn. Many of these genes also improve freezing tolerance(or resistance); the physical stresses incurred during freezing anddrought are similar in nature and may be mitigated in similar fashion.Benefit may be conferred via constitutive expression of these genes, butthe preferred means of expressing these novel genes may be through theuse of a turgor-induced promoter (such as the promoters for theturgor-induced genes described in Guerrero et al., 1987 and Shagan etal., 1993 which are incorporated herein by reference). Spatial andtemporal expression patterns of these genes may enable corn to betterwithstand stress.

It is proposed that expression of genes that are involved with specificmorphological traits that allow for increased water extractions fromdrying soil would be of benefit. For example, introduction andexpression of genes that alter root characteristics may enhance wateruptake. It is also contemplated that expression of genes that enhancereproductive fitness during times of stress would be of significantvalue. For example, expression of genes that improve the synchrony ofpollen shed and receptiveness of the femal flower parts, i.e., silks,would be of benefit. In addition it is proposed that expression of genesthat minimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value.

Given the overall role of water in determining yield, it is contemplatedthat enabling corn to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of corn to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

4. Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into monocotyledonous plants such asmaize. It is possible to produce resistance to diseases caused byviruses, bacteria, fungi and nematodes. It is also contemplated thatcontrol of mycotoxin producing organisms may be realized throughexpression of introduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay impart resistance to said virus. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit said replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes may also increase resistance to viruses. Further it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called "peptide antibiotics,"pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in monocotyledonousplants such as maize may be useful in conferring resistance to bacterialdisease. These genes are induced following pathogen attack on a hostplant and have been divided into at least five classes of proteins (Bol,Linthorst, and Cornelissen, 1990). Included amongst the PR proteins areβ-1, 3-glucanases, chitinases, and osmotin and other proteins that arebelieved to function in plant resistance to disease organisms. Othergenes have been identified that have antifungal properties, e.g., UDA(stinging nettle lectin) and hevein (Broakaert et al., 1989;Barkai-Golan et al., 1978). It is known that certain plant diseases arecaused by the production of phytotoxins. It is proposed that resistanceto these diseases would be achieved through expression of a novel genethat encodes an enzyme capable of degrading or otherwise inactivatingthe phytotoxin. It is also contemplated that expression novel genes thatalter the interactions between the host plant and pathogen may be usefulin reducing the ability the disease organism to invade the tissues ofthe host plant, e.g., an increase in the waxiness of the leaf cuticle orother morphological characteristics.

Plant parasitic nematodes are a cause of disease in many plants,including maize. It is proposed that it would be possible to make thecorn plant resistant to these organisms through the expression of novelgenes. It is anticipated that control of nematode infestations would beaccomplished by altering the ability of the nematode to recognize orattach to a host plant and/or enabling the plant to produce nematicidalcompounds, including but not limited to proteins.

5. Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with monocotyledonous plants such as maize is a significantfactor in rendering the grain not useful. These fungal organisms do notcause disease symptoms and/or interfere with the growth of the plant,but they produce chemicals (mycotoxins) that are toxic to animals. It iscontemplated that inhibition of the growth of these fungi would bereduce the synthesis of these toxic substances and therefore reducegrain losses due to mycotoxin contamination. It is also proposed that itmay be possible to introduce novel genes into monocotyledonous plantssuch as maize that would inhibit synthesis of the mycotoxin withoutinterfering with fungal growth. Further, it is contemplated thatexpression of a novel gene which encodes an enzyme capable of renderingthe mycotoxin nontoxic would be useful in order to achieve reducedmycotoxin contamination of grain. The result of any of the abovemechanisms would be a reduced presence of mycotoxins on grain.

6. Grain Composition or Quality

Genes may be introduced into monocotyledonous plants, particularlycommercially important cereals such as maize, to improve the grain forwhich the cereal is primarily grown. A wide range of novel transgenicplants produced in this manner may be envisioned depending on theparticular end use of the grain.

The largest use of maize grain is for feed or food. Introduction ofgenes that alter the composition of the grain may greatly enhance thefeed or food value. The primary components of maize grain are starch,protein, and oil. Each of these primary components of maize grain may beimproved by altering its level or composition. Several examples may bementioned for illustrative purposes but in no way provide an exhaustivelist of possibilities.

The protein of cereal grains including maize is suboptimal for feed andfood purposes especially when fed to pigs, poultry, and humans. Theprotein is deficient in several amino acids that are essential in thediet of these species, requiring the addition of supplements to thegrain. Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after corn is supplemented with other inputs forfeed formulations. For example, when corn is supplemented with soybeanmeal to meet lysine requirements methionine becomes limiting. The levelsof these essential amino acids in seeds and grain may be elevated bymechanisms which include, but are not limited to, the introduction ofgenes to increase the biosynthesis of the amino acids, decrease thedegradation of the amino acids, increase the storage of the amino acidsin proteins, or increase transport of the amino acids to the seeds orgrain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.Examples may include the introduction of DNA that decreases theexpression of members of the zein family of storage proteins. This DNAmay encode ribozymes or antisense sequences directed to impairingexpression of zein proteins or expression of regulators of zeinexpression such as the opaque-2 gene product. It is also proposed thatthe protein composition of the grain may be modified through thephenomenon of cosupression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al.,1991). Additionally, the introduced DNA may encode enzymes which degradezeins. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable-energy-content and--density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, β-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Genes may be introducedthat alter the balance of fatty acids present in the oil providing amore healthful or nutritive feedstuff. The introduced DNA may alsoencode sequences that block expression of enzymes involved in fatty acidbiosynthesis, altering the proportions of fatty acids present in thegrain such as described below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising primarily maize or other cereal grains possessesinsufficient quantitities of vitamins and must be supplemented toprovide adequate nutritive value. Introduction of genes that enhancevitamin biosynthesis in seeds may be envisioned including, for example,vitamins A, E, B₁₂, choline, and the like. Maize grain also does notpossess sufficient mineral content for optimal nutritive value. Genesthat affect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of maize or other cereals forfeed and food purposes might be described. The improvements may not evennecessarily involve the grain, but may, for example, improve the valueof the corn for silage. Introduction of DNA to accomplish this mightinclude sequences that alter lignin production such as those that resultin the "brown midrib" phenotype associated with superior feed value forcattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of corn and improve the valueof the products resulting from the processing. The primary method ofprocessing corn is via wetmilling. Maize may be improved though theexpression of novel genes that increase the efficiency and reduce thecost of processing such as by decreasing steeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Futhermore, it may be worthwhile tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moities of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn, the value of which may beimproved by introduction and expression of genes. The quantity of oilthat can be extracted by wetmilling may be elevated by approaches asdescribed for feed and food above. Oil properties may also be altered toimprove its performance in the production and use of cooking oil,shortenings, lubricants or other oil-derived products or improvement ofits health attributes when used in the food-related applications. Novelfatty acids may also be synthesized which upon extraction can serve asstarting materials for chemical syntheses. The changes in oil propertiesmay be achieved by altering the type, level, or lipid arrangement of thefatty acids present in the oil. This in turn may be accomplished by theaddition of genes that encode enzymes that catalyze the synthesis ofnovel fatty acids and the lipids possessing them or by increasing levelsof native fatty acids while possibly reducing levels of precursors.Alternatively DNA sequences may be introduced which slow or block stepsin fatty acid biosynthesis resulting in the increase in precursor fattyacid intermediates. Genes that might be added include desaturases,epoxidases, hydratases, dehydratases, and other enzymes that catalyzereactions involving fatty acid intermediates. Representative examples ofcatalytic steps that might be blocked include the desaturations fromstearic to oleic acid and oleic to linolenic acid resulting in therespective accumulations of stearic and oleic acids. Another example isthe blockage of elongation steps resulting in the accumulation of c₈ toc₁₂ saturated fatty acids.

Improvements in the other major corn wetmilling products, corn glutenmeal and corn gluten feed, may also be achieved by the introduction ofgenes to obtain novel corn plants. Representative possibilities includebut are not limited to those described above for improvement of food andfeed value.

In addition it may further be considered that the corn plant be used forthe production or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in thecorn plant previously. The novel corn plants producing these compoundsare made possible by the introduction and expression of genes by corntransformation methods. The vast array of possibilities include but arenot limited to any biological compound which is presently produced byany organism such as proteins, nucleic acids, primary and intermediarymetabolites, carbohydrate polymers, etc. The compounds may be producedby the plant, extracted upon harvest and/or processing, and used for anypresently recognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance γ-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

7. Plant Agronomic Characteristics

Two of the factors determining where corn can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow corn,there are varying limitations on the maximal time it is allowed to growto maturity and be harvested. The corn to be grown in a particular areais selected for its ability to mature and dry down to harvestablemoisture content within the required period of time with maximumpossible yield. Therefore, corn of varying maturities is developed fordifferent growing locations. Apart from the need to dry downsufficiently to permit harvest is the desirability of having maximaldrying take place in the field to minimize the amount of energy requiredfor additional drying post-harvest. Also the more readily the grain candry down, the more time there is available for growth and kernel fill.It is considered that genes that influence maturity and/or dry down canbe identified and introduced into corn lines using transformationtechniques to create new corn varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in corn.

It is contemplated that genes may be introduced into corn that wouldimprove standability and other plant growth characteristics. Expressionof novel genes which confer stronger stalks, improved root systems, orprevent or reduce ear droppage would be of great value to the farmer. Itis proposed that introduction and expression of genes that increase thetotal amount of photoassimilate available by, for example, increasinglight distribution and/or interception would be advantageous. Inaddition the expression of genes that increase the efficiency ofphotosynthesis and/or the leaf canopy would further increase gains inproductivity. Such approaches would allow for increased plantpopulations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. It is proposed thatoverexpression of genes within corn that are associated with "staygreen" or the expression of any gene that delays senescence wouldachieve be advantageous. For example, a nonyellowing mutant has beenidentified in Festuca pratensis (Davies et al., 1990). Expression ofthis gene as well as others may prevent premature breakdown ofchlorophyll and thus maintain canopy function.

8. Nutrient Utilization

The ability to utilize available nutrients may be a limiting factor ingrowth of monocotyledonous plants such as maize. It is proposed that itwould be possible to alter nutrient uptake, tolerate pH extremes,mobilization through the plant, storage pools, and availability formetabolic activities by the introduction of novel genes. Thesemodifications would allow a plant such as maize to more efficientlyutilize available nutrients. It is contemplated that an increase in theactivity of, for example, an enzyme that is normally present in theplant and involved in nutrient utilization would increase theavailability of a nutrient. An example of such an enzyme would bephytase. It is also contemplated that expression of a novel gene maymake a nutrient source available that was previously not accessible,e.g., an enzyme that releases a component of nutrient value from a morecomplex molecule, perhaps a macromolecule.

9. Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al, 1990).

A number of mutations were discovered in maize that confer cytoplasmicmale sterility. One mutation in particular, referred to as T cytoplasm,also correlates with sensitivity to Southern corn leaf blight. A DNAsequence, designated TURF-13 (Levings, 1990), was identified thatcorrelates with T cytoplasm. It is proposed that it would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production itis proposed that genes encoding restoration of male fertility may alsobe introduced.

10. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. It is contemplatedthat when two or more genes are introduced together by cotransformationthat the genes will be linked together on the host chromosome. Forexample, a gene encoding a Bt gene that confers insect resistance on theplant may be introduced into a plant together with a bar gene that isuseful as a selectable marker and confers resistance to the herbicideIgnite® on the plant. However, it may not be desirable to have an insectresistant plant that is also resistant to the herbicide Ignite®. It isproposed that one could also introduce an antisense bar gene that isexpressed in those tissues where one does not want expression of the bargene, e.g., in whole plant parts. Hence, although the bar gene isexpressed and is useful as a selectable marker, it is not useful toconfer herbicide resistance on the whole plant. The bar antisense geneis a negative selectable marker.

It is also contemplated that a negative selection is necessary in orderto screen a population of transformants for rare homologous recombinantsgenerated through gene targeting. For example, a homologous recombinantmay be identified through the inactivation of a gene that was previouslyexpressed in that cell. The antisense gene to neomycinphosphotransferase II (nptII) has been investigated as a negativeselectable marker in tobacco (Nicotiana tabacum) and Arabidopsisthaliana (Xiang, C. and Guerra, D. J. 1993). In this example both senseand antisense npt II genes are introduced into a plant throughtransformation and the resultant plants are sensitive to the antibiotickanamycin. An introduced gene that integrates into the host cellchromosome at the site of the antisense nptII gene, and inactivates theantisense gene, will make the plant resistant to kanamycin and otheraminoglycoside antibiotics. Therefore, rare site specific recombinantsmay be identified by screening for antibiotic resistance. Similarly, anygene, native to the plant or introduced through transformation, thatwhen inactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers may also be usefulin other ways. One application is to contruct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, J., 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluoruracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thethe cytosine deaminase gene through genetic segregation of thetransposable element and the cytosine deaminase gene. Other genes thatencode proteins that render the plant sensitive to a certain compoundwill also be useful in this context. For example, T-DNA gene 2 fromAgrobacterium tumefaciens encodes a protein that catalyzes the.conversion of α-naphthalene acetamide (NAM) to α-napthalene acetic acid(NAA) renders plant cells sensitive to high concentrations of NAM(Depicker et al., 1988).

It is also contemplated that negative selectable markers may be usefulin the contruction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stabily integrated into the genome.It is proposed that this would be desirable, for example, when transientexpression of the autonomous element is desired to activate in trans thetransposition of a defective transposable element, such as Ds, butstable integration of the autonomous element is not desired. Thepresence of the autonomous element may not be desired in order tostabilize the defective element, i.e., prevent it from furthertransposing. However, it is proposed that if stable integration of anautonomous transposable element is desired in a plant the presence of anegative selectable marker may make it possible to eliminate theautonomous element during the breeding process.

D. Non-Protein-Expressing Sequences

1. RNA-Expressing

DNA may be introduced into corn and other monocots for the purpose ofexpressing RNA transcripts that function to affect plant phenotype yetare not translated into protein. Two examples are antisense RNA and RNAwith ribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby a mechanism of cosuppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al, 1.991; Smith et al., 1990; Napoli, C.et al., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

2. Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be inserted into a gene and cause mutations. TheseDNA elements may be inserted in order to inactivate (or activate) a geneand thereby "tag" a particular trait. In this instance the transposableelement does not cause instability of the tagged mutation, because theutility of the element does not depend on its ability to move in thegenome. Once a desired trait is tagged, the introduced DNA sequence maybe used to clone the corresponding gene, e.g., using the introduced DNAsequence as a PCR primer together with PCR gene cloning techniques(Shapiro, 1983; Dellaporta et al., 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary"labels" of those cells and plants and seeds thereof. It would not benecessary for a label DNA,element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labelled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief,1989), which can be positioned around an expressible gene of interest toeffect an increase in overall expression of the gene and diminishposition dependant effects upon incorporation into the plant genome(Stief et al., 1989; Phi-Van et al., 1990).

III. DNA Delivery

Following the generation of recipient cells, the present inventiongenerally next includes steps directed to introducing an exogenous DNAsegment, such as a cDNA or gene, into a recipient cell to create atransformed cell. The frequency of occurrence of cells receiving DNA isbelieved to be low. Moreover, it is most likely that not all recipientcells receiving DNA segments will result in a transformed cell whereinthe DNA is stably integrated into the plant genome and/or expressed.Some may show only initial and transient gene expression. However,certain cells from virtually any monocot species may be stablytransformed, and these cells developed into transgenic plants, throughthe application of the techniques disclosed herein.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

A. Electroporation

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Ser. No. 07/635,279filed Dec. 28, 1990, incorporated herein by reference) will beparticularly advantageous. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding.

To effect transformation by electroporation one may employ eitherfriable tissues such as a suspension culture of cells, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. One would partially degrade the cell wallsof the chosen cells by exposing them to pectin-degrading enzymes(pectolyases) or mechanically wounding in a controlled manner. Suchcells would then be recipient to DNA transfer by electroporation, whichmay be carried out at this stage, and transformed cells. then identifiedby a suitable selection or screening protocol dependent on the nature ofthe newly incorporated DNA.

B. Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. In an illustrative embodiment,non-embryogenic BMS cells were bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucoronidase or bar gene engineered for expression inmaize. Bacteria were inactivated by ethanol dehydration prior tobombardment. A low level of transient expression of the β-glucoronidasegene was observed 24-48 hours following DNA delivery. In addition,stable transformants containing the bar gene were recovered followingbombardment with either E. coli or Agrobacterium tumefaciens cells. Itis contemplated that particles may contain DNA rather than be coatedwith DNA. Hence it is proposed that DNA-coated particles may increasethe level of DNA delivery via particle bombardment but are not, in andof themselves, necessary.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. Results from such small scale optimizationstudies are disclosed herein and the execution of other routineadjustments will be known to those of skill in the art in light of thepresent disclosure.

IV. Production and Characterization of Stable Transgenic Corn

After effecting delivery of exogenous DNA to recipient cells by any ofthe methods discussed above, the next steps of the invention generallyconcern identifying the transformed cells for further culturing andplant regeneration. As mentioned above, in order to improve the abilityto identify transformants, one may desire to employ a selectable orscreenable marker gene as, or in addition to, the expressible gene ofinterest. In this case, one would then generally assay the potentiallytransformed cell population by exposing the cells to a selective agentor agents, or one would screen the cells for the desired marker genetrait.

A. Selection

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility in the practice of the invention. Tissue can be placed on anyporous, inert, solid or semi-solid support for bombardment, includingbut not limited to filters and solid culture medium. Bialaphos andglyphosate are provided as examples of agents suitable for selection oftransformants, but the technique of this invention is not limited tothem.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or x-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. In anillustrative embodiment embryogenic type II callus of Zea mays L. wasselected with sub-lethal levels of bialaphos. Slowly growing tissue wassubsequently screened for expression of the luciferase gene andtransformants were identified. In this example, neither selection norscreening conditions employed were sufficient in and of themselves toidentify transformants. Therefore it is proposed that combinations ofselection and screening will enable one to identify transformants in awider variety of cell and tissue types.

B. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, theinventors have modified MS and N6 media (see Table 1) by includingfurther substances such as growth regulators. A preferred growthregulator for such purposes is dicamba or 2,4-D. However, other growthregulators may be employed, including NAA, NAA+2,4-D or perhaps evenpicloram. Media improvement in these and like ways was found tofacilitate the growth of cells at specific developmental stages. Tissueis preferably maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least two weeks, thentransferred to media conducive to maturation of embryoids. Cultures aretransferred every two weeks on this medium. Shoot development willsignal the time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoilless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber at about 85% relative humidity, 600 ppm CO₂, and25-250 microeinsteins m⁻².s⁻¹ of light. Plants are preferably. maturedeither in a growth chamber or greenhouse. Plants are regenerated fromabout 6 weeks to 10 months after a transformant is identified, dependingon the initial tissue. During regeneration, cells are grown on solidmedia in tissue culture vessels. Illustrative embodiments of suchvessels are petri dishes and Plant Con®s. Regenerating plants arepreferably grown at about 19 to 28° C. After the regenerating plantshave reached the stage of shoot and root development, they may betransferred to a greenhouse for further growth and testing.

In one study, R₀ plants were regenerated from transformants of anA188×B73 suspension culture line (SC82), and these plants exhibited aphenotype expected of the genotype of hybrid A188×B73 from which thecallus and culture were derived. The plants were similar in height toseed-derived A188 plants (3-5 ft tall) but had B73 traits such asanthocyanin accumulation in stalks and prop roots, and the presence ofupright leaves. It would also be expected that some traits in thetransformed plants would differ from their source, and indeed somevariation will likely occur.

In an exemplary embodiment, the proportion of regenerating plantsderived from transformed callus that successfully grew and reachedmaturity after transfer to the greenhouse was 97% (73 of 76). R₀ plantsin the greenhouse are tested for fertility by backcrossing thetransformed plants with seed-derived plants by pollinating the R₀ earswith pollen from seed derived inbred plants and this resulted in kerneldevelopment. In addition, pollen was collected from R₀ plants and usedto pollinate seed derived inbred plants, resulting in kerneldevelopment. Although fertility can vary from plant to plant greaterthan 100 viable progeny can be routinely recovered from each transformedplant through use of both the ear and pollen for doing crosses.

Note, however, that occasionally kernels on transformed plants mayrequire embryo rescue due to cessation of kernel development andpremature senescence of plants. To rescue developing embryos, they areexcised from surface-disinfected kernels 10-20 days post-pollination andcultured. An embodiment of media used for culture at this stagecomprises MS salts, 2% sucrose, and 5.5 g/l agarose. In an illustrativeembodiment of embryo rescue, large embryos (defined as greater than 3 mmin length) are germinated directly on an appropriate media. Embryossmaller than that were cultured for one week on media containing theabove ingredients along with 10⁻⁵ M abscisic acid and then transferredto growth regulator-free medium for germination.

Progeny may be recovered from the transformed plants and tested forexpression of the exogenous expressible gene by localized application ofan appropriate substrate to plant parts such as leaves. In the case ofbar transformed plants, it was found that transformed parental plants(R₀) and their progeny (R₁) exhibited no bialaphos-related necrosisafter localized application of the herbicide Basta® to leaves, if therewas functional PAT activity in the plants as assessed by an in vitroenzymatic assay. In one study, of 28 progeny (R₁) plants tested, 50%(N=14) had PAT activity. All PAT positive progeny tested contained bar,confirming that the presence of the enzyme and the resistance tobialaphos were associated with the transmission through the germline ofthe marker gene.

C. Characterization

To confirm the presence of the exogenous DNA or "transgene (s)" in theregenerating plants, a variety of assays may be performed. Such assaysinclude, for example, "molecular biological" assays, such as Southernand Northern blotting and PCR; "biochemical" assays, such as detectingthe presence of a protein product, e.g., by immunological means (ELISAsand Western blots) or by enzymatic function; plant part assays, such asleaf or root assays; and also, by analyzing the phenotype of the wholeregenerated plant.

1. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from callus cell lines or any plant parts todetermine the presence of the exogenous gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of DNA elements introduced through the methods of thisinvention may be determined by polymerase chain reaction (PCR). Usingthis technique discreet fragments of DNA are amplified and detected bygel electrophoresis. This type of analysis permits one to determinewhether a gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome. It is theexperience of the inventors, however, that DNA has been integrated intothe genome of all transformants that demonstrate the presence of thegene through PCR analysis. In addition, it is not possible using PCRtechniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR e.g., thepresence of a gene, but also demonstrates integration into the genomeand characterizes each individual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a gene.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a transgene to progeny. It is the experienceof the inventors that in most instances the characteristic Southernhybridization pattern for a given transformant will segregate in progenyas one or more Mendelian genes (Spencer et al., 1992; Spencer et al, inpress) indicating stable inheritance of the transgene. For example, inone study, of 28 progeny (R₁) plants tested, 50% (N=14) contained bar,confirming transmission through the germline of the marker gene. Thenonchimeric nature of the callus and the parental transformants (R₀) wassuggested by germline transmission and the identical Southern blothybridization patterns and intensities of the transforming DNA incallus, R₀ plants and R₁ progeny that segregated for the transformedgene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA will only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced genes. In this applicationof PCR it is first necessary to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then through the use ofconventional PCR techniques amplify the DNA. In most instances PCRtechniques, while useful, will not demonstrate integrity of the RNAproduct. Further information about the nature of the RNA product may beobtained by Northern blotting. This technique will demonstrate thepresence of an RNA species and give information about the integrity ofthat RNA. The presence or absence of an RNA species can also bedetermined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

2. Gene Expression

While Southern blotting and PCR may be used to detect the gene(s) inquestion, they do not provide information as to whether the gene isbeing expressed. Expression may be evaluated by specifically identifyingthe protein products of the introduced genes or evaluating thephenotypic changes brought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focussing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabelled acetylated phosphinothricin fromphosphinothricin and ¹⁴ C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may beanalyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays. An example is to evaluate resistance toinsect feeding.

D. Establishment of the Introduced DNA in Other Maize Varieties

Fertile, transgenic plants may then be used in a conventional maizebreeding program in order to incorporate the introduced DNA into thedesired lines or varieties. Methods and references for convergentimprovement of corn are given by Hallauer et al., (1988) incorporatedherein by reference. Among the approaches that conventional breedingprograms employ is a conversion process (backcrossing). Briefly,conversion is performed by crossing the initial transgenic ferile plantto elite inbred lines. The progeny from this cross will segregate suchthat some of the plants will carry the recombinant DNA whereas some willnot. The plants that do not carry the DNA are then crossed again to theelite inbred lines resulting in progeny which segregate once more. Thisbackcrossing process is repeated until the original elite inbred hasbeen converted to a line containing the recombinant DNA, yet possessingall important attributed originally found in the parent. Generally, thiswill require about 6-8 generations. A separate backcrossing program willbe generally used for every elite line that is to be converted to agenetically engineered elite line.

Generally, the commercial value of the transformed corn produced hereinwill be greatest if the recombinant DNA can be incorporated into manydifferent hybrid combinations. A farmer typically grows several hybridsbased on differences in maturity, standability, and other agronomictraits. Also, the farmer must select a hybrid based upon his or hergeographic location since hybrids adapted to one region are generallynot adapted to another because of differences in such traits asmaturity, disease, and insect resistance. As such, it is necessary toincorporate the introduced DNA into a large number of parental lines sothat many hybrid combinations can be produced containing the desirableDNA.

Corn breeding and the techniques and skills required to transfer genesfrom one line or variety to another are well known to those skilled inthe art. Thus, introducing recombinant DNA into any other line orvariety can be accomplished by these breeding procedures.

E. Uses of Transgenic Plants

The transgenic plants produced herein are expected to be useful for avariety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as pest resistance, herbicideresistance or increased yield), beneficial to the consumer of the grainharvested from the plant (e.g., improved nutritive content in human foodor animal feed), or beneficial to the food processor (e.g., improvedprocessing traits). In such uses, the plants are generally grown for theuse of their grain in human or animal foods. However, other parts of theplants, including stalks, husks, vegetative parts, and the like, mayalso have utility, including use as part of animal silage or forornamental purposes. Often, chemical constitutents (e.g., oils orstarches) of corn and other crops are extracted for foods or industrialuse and transgenic plants may be created which have enhanced or modifiedlevels of such components.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the recombinant DNA may be transferred, e.g.,from corn cells to cells of other species, e.g., by protoplast fusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique "signature sequences" orother marker sequences which can be used to identify proprietary linesor varieties.

The inventors have been successful in producing fertile transgenicmonocot plants (maize) where others have failed. Aspects of the methodsof the present invention for producing the fertile, transgenic cornplants comprise, but are not limited to, isolation of recipient cellsusing media conducive to specific growth patterns, choice of selectivesystems that permit efficient detection of transformation; modificationsof DNA delivery methods to introduce genetic vectors with exogenous DNAinto cells; invention of methods to regenerate plants from transformedcells at a high frequency; and the production of fertile transgenicplants capable of surviving and reproducing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of plasmids (vectors) used inbombardment experiments.

FIG. 1(A). Schematic representation of the expression cassette ofpDPG165 containing the bar gene.

FIG. 1(B). Schematic representation of the expression cassette ofpDPG208 containing the uidA gene encoding β-glucoronidase (GUS).

FIG. 1(C). Map of plasmid pDPG165 containing the bar gene.

FIG. 1(D) Map of plasmid pDPG208 containing the uidA gene.

FIG. 1(E) Map of plasmid pAGUS1, also known as pDPG141, in which the5'-noncoding and 5'-coding sequences wre modified to incorporate theKozak consensus sequence and HindIII restriction site. The nucleotidesequence is seq id no:2.

FIG. 1(F). Restriction map of the plasmid pDPG237 containing the Sn:bol3cDNA.

FIG. 1(G). Map of plasmid pDPG232 incorporating the Rsn cDNA with a 35Spromoter and Tr 7 3' end.

FIG. 1(H). Map of plasmid pDPG313 containing the aroA gene and the35S-histone fusion promoter in addition to the bar expression cassette.

FIG. 1(I). Map of plasmid pDPG314 containing the aroA gene and the35S-histone fusion promoter in addition to the bar expression cassette.

FIG. 1(J). Map of plasmid pDPG315 containing the aroA gene and thehistone fusion promoter in addition to the bar expression cassette.

FIG. 1(K). Map of plasmid pDPG316 containing the aroA gene and thehistone fusion promoter in addition to the bar expression cassette.

FIG. 1(L). Map of plasmid pDPG317 containing the aroA gene and the35S-histone fusion promoter in addition to the bar expression cassette.

FIG. 1(M). Map of plasmid pDPG318 containing the aroA gene and theα-tubulin promoter in addition to the bar expression cassette.

FIG. 1(N). Map of plasmid pDPG319 containing the aroA gene and theα-tubulin promoter in addition to the bar expression cassette.

FIG. 1(O). Map of plasmid pDPG290 containing the B. thuringiensiscrystal toxin protein gene Iab6 with a 35S promoter.

FIG. 1(P). Map of plasmid pDPG300 containing containing the B.thuringiensis crystal toxin protein gene Iab6 with a 35S promoter inaddition to the bar expression cassette from pDPG165.

FIG. 1(Q). Map of plasmid pDPG301 containing containing the B.thuringiensis crystal toxin protein gene Iab6 with a 35S promoter inaddition to the bar expression cassette from pDPG165.

FIG. 1(R). Map of plasmid pDPG302 containing containing, the B.thuringiensis crystal toxin protein gene Iab6 with a 35S promoter inaddition to the bar expression cassette from pDPG165.

FIG. 1(S). Map of plasmid pDPG303 containing containing the B.thuringiensis crystal toxin protein gene Iab6 with a 35S promoter inaddition to the bar expression cassette from pDPG165.

FIG. 1(T). Map of plasmid pDPG386, a plasmid containing the wheat dwarfvirus replicon and containing a neomycin phosphotransferase II gene.This virus replicates in plant cells as well as bacteria.

FIG. 1(U). Map of plasmid pDPG387, a plasmid containing the wheat dwarfvirus replicon and containing a neomycin phosphotransferase II gene andthe uidA gene encoding GUS. This virus replicates in plant cells as wellas bacteria.

FIG. 1(V). Map of plasmid pDPG388, a plasmid containing the wheat dwarfvirus replicon and containing a neomycin phosphotransferase II gene andthe bar. This virus replicates in plant cells as well as bacteria.

FIG. 1(W). Map of plasmid pDPG389, a plasmid containing the wheat dwarfvirus replicon and containing a neomycin phosphotransferase II gene andthe bar gene. This virus replicates in plant cells as well as bacteria.

FIG. 1(X). Map of plasmid pDPG140.

FIG. 1(Y). Map of plasmid pDPG172 containing the luciferase gene and themaize alcohol dehydrogenase I promoter and intron one.

FIG. 1(Z). Map of plasmid pDPG425 containing a maize EPSPS gene mutatedto confer resistance to glyphosate.

FIG. 1(AA). Map of plasmid pDPG427 containing a maize EPSPS gene mutatedto confer resistance to glyphosate.

FIG. 1(BB). Map of plasmid pDPG451 containing the 35S promoter--adhintron- mtlD- Tr7 expression cassette. Expression of this cassette willlead to accumulation of mannitol in the cells.

FIG. 1(CC). Map of plasmid pDPG354 containing a synthetic Bt gene (seeFIG. 12).

FIG. 1(DD). Map of plasmid pDPG344 containing the proteinase inhibitorII gene from tomato.

FIG. 1(EE). Map of plasmid pDPG337 containing a synthetic Bt gene (seeFIG. 12).

FIG. 2. Appearance of cell colonies which emerge on selection plateswith bialaphos. Such colonies appear 6-7 weeks after bombardment.

FIG. 2(A) SC82 bialaphos-resistant colony selected on 1 mg/l bialaphos.

FIG. 2(B) Embryogenic SC82 bialaphos-resistant callus selected andmaintained on 1 mg/l bialaphos.

FIG. 3. Phosphinothricin acetyl transferase (PAT) activity inembryogenic SC82 callus transformants designated E1-E11 and anonselected control (E0). 25 μg of protein extract were loaded per lane.B13 is a BMS-bar transformant. BMS is Black Mexican Sweet corn.Activities of the different transformants varied approximately 10 foldbased on the intensities of the bands.

FIG. 4. Integration of the bar gene in bialaphos-resistant SC82 callusisolates E1-E11. DNA gel blot of genomic DNA (4 μg/digest) from E1-E11and a nonselected control (E0) digested with EcoRI and HindIII. Themolecular weights in kb are shown on the left and right. The blot washybridized with ³² P-labeled bar from pDPG165 (˜25×10⁶ Cerenkov cpm).Lanes designated 1 and 5 copies refer to the diploid genome and contain1.9 and 9.5 pg respectively of the 1.9 kb bar expression unit releasedfrom pDPG165 with EcoRI and HindIII.

FIG. 5. Integration of exogenous genes in bialaphos-resistant SC716isolates R1-R21.

FIG. 5(A) DNA gel blot of genomic DNA (6 μg/digest) from transformantsisolated from suspension culture of A188×B73 (SC716), designated R1-R21,were digested with EcoRI and HindIII and hybridized to ³² P-labeled barprobe (˜10×10⁶ Cerenkov cpm). Molecular weight markers in kb are shownon the left and right. Two copies of the bar expression unit per diploidgenome is 5.7 pg of the 1.9 kb EcoRI/Hind fragment from pDPG165.

FIG. 5(B) The blot from A was washed and hybridized with ³² P-labelledGUS probe (˜35×10⁵ Cerenkov cpm). Two copies of the 2.1 kbGUS-containing EcoRI/HindIII fragment from pDPG208 is 6.3 pg.

FIG. 6. Histochemical determination of GUS activity in bar-transformedSC82 callus line Y13. This bialaphos-resistant callus line, Y13, whichcontained intact GUS coding sequences was tested for GUS activity threemonths post-bombardment. In this figure, differential staining of thecallus was observed.

FIG. 7. Mature R₀ Plant, Developing Kernels and Progeny.

FIG. 7(A). Mature transgenic R₀ plant regenerated from an E2/E5 callus.

FIG. 7(B) Progeny derived from an E2/E5 plant by embryo rescue;segregant bearing the resistance gene on the right, and lacking the geneon the left.

FIG. 7(C) Using pollen from transformed R₁ plants to pollinate B73 ears,large numbers of seed have been recovered.

FIG. 7(D) A transformed ear from an R₁ plant crossed with pollen from anon-transformed inbred plant.

FIG. 8. Functional Expression of Introduced Genes in Transformed R₀ andR₁ Plants.

FIG. 8(A) Basta^(R) resistance in transformed R₀ plants. A Basta^(R)solution was applied to a large area (about 4×8 cm) in the center ofleaves of nontransformed A188×B73 plant (left) and a transgenic R₀E3/E4/E6 plant (right).

FIG. 8(B) Basta^(R) resistance in transformed R₁ plants. Basta^(R) wasalso applied to leaves of four R₁ plants; two plants without bar (left)and two plants containing bar (right). The herbicide was applied to R₁plants in 1 cm circles to four locations on each leaf, two on each sideof the midrib. Photographs were taken six days after application.

FIG. 8(C) GUS activity in leaf tissue of a transgenic R₀ plant.Histochemical determination of GUS activity in leaf tissue of a plantregenerated from cotransformed callus line Y13 (right) and anontransformed tissue culture derived plant (left). Bar=1 cm.

FIG. 8(D) Light micrograph of the leaf segment from a Y13 plant shown in(C), observed in surface view under bright field optics. GUS activitywas observed in many cell types throughout the leaf tissue(magnification=230×). (E) Light micrograph as in (D) of control leaf.

FIG. 9. PAT Activity in Protein Extracts of R₀ Plants. Extracts from oneplant derived from each of the four transformed regenerable callus linesfrom a suspension culture of A188×B73, SC82 (E10, E11, E2/E5, andE3/E4/E6) were tested for PAT activity (The designations E2/E5 andE3/E4/E6 represent transformed cell lines with identical DNA gel blothybridization patterns; the isolates were most likely separated duringthe culturing and selection process.) Protein extracts from anontransformed B73 plant and a Black Mexican Sweet (BMS) cell culturebar transformant were included as controls. Approximately 50 microgramsof total protein was used per reaction.

FIG. 10. DNA Gel Blot Analysis of Genomic DNA from Transformed Callusand Corresponding R₀ Plants Probed with bar. Genomic DNA was digestedwith EcoRI and HindIII, which released the 1.9 kb bar expression unit(CaMV 35S promoter-bar-Tr7 3'-end) from pDPG165, the plasmid used formicroprojectile bombardment transformation of SC82 cells, and hybridizedto bar. The molecular weights in kb are shown on the left and right.Lanes designated E3/E4/E6, E11, E2/E5, and E10 contained 5 μg of eithercallus (C) or R₀ plant DNA. The control lane contained DNA from anontransformed A188×B73 plant. The lane designated "1 copy" contained2.3 pg of the 1.9 kb EcoRI/HindIII fragment from pDPG165 representingone copy per diploid genome.

FIG. 11. PAT Activity and DNA Gel Blot Analysis of Segregating Progenyof E2/E5 R₀ Plants.

FIG. 11(A) Analysis of PAT activity in ten progeny (lanes a-j) and anontransformed control plant (lane k). Lanes designated a, b-h, i, and jcontained protein extracts from progeny of separate parental R₀ plants.The lane designated callus contained protein extract from E2/E5 callus.Approximately 25 micrograms of total protein were used per reaction.

FIG. 11(B) DNA gel blot analysis of genomic DNA isolated from the tenprogeny analyzed in A. Genomic DNA (5 μg/lane) was digested with SmaI,which releases a 0.6 kb fragment containing bar from pDPG165, andhybridized with bar probe. The lane designated R₀ contained DNA from theR₀ parent of progeny a. The lane designated 1 copy contained pDPG165digested with SmaI to represent approximately 1 copy of the 0.6 kbfragment per diploid genome (0.8 pg).

FIG. 12 DNA sequence of a synthetic Bt gene coding for the toxin portionof the endotoxin protein produced by Bacillus thuringiensis subsp.kurstaki strain HD73 (M. J. Adang et al. 1985). This gene wassynthesized and assembled using standard techniques to contain codonsthat are more preferred for translation in maize cells. A translationstop codon was introduced after the 613th codon to terminate thetranslation and allow synthesis of a Bt endotoxin protein consisting ofthe first 613 amino acids (including, the f-met) of the Bt protein. Thenucleic acid sequence is represented by seq id no:5 and the amino acidsequence by seq id no:6.

FIG. 13 DNA sequence of a synthetic Bt gene coding for the toxin portionof the endotoxin protein produced by Bacillus thuringiensis strain HD1.This gene was synthesized and assembled using standard techniques tocontain codons that are more preferred for translation in maize cells.The nucleic acid sequence is represented by seq id no:7 and the aminoacid sequence by seq id no:8.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the first time, fertile transgenic maize plants have been produced,opening the door to new vistas of crop improvement based on in vitrogenetic transformation. The inventors have succeeded where others havefailed by combining and modifying numerous steps in the overall processleading from somatic cell to transgenic plant. Although the methodsdisclosed herein are part of a unified process, for illustrativepurposes they may be subdivided into: culturing cells to be recipientsfor exogenous DNA; cryopreserving recipient cells; constructing vectorsto deliver the DNA to cells; delivering DNA to cells; assaying forsuccessful transformations; using selective agents if necessary toisolate stable transformants; regenerating plants from transformants;assaying those plants for gene expression and for identification of theexogenous DNA sequences; determining whether the transgenic plants arefertile; and producing offspring of the transgenic plants. The inventionalso relates to transformed maize cells, transgenic plants and pollenproduced by said plants.

I. Recipient Cells

Tissue culture requires media and controlled environments. "Media"refers to the numerous nutrient mixtures that are used to grow cells invitro, that is, outside of the intact living organism. The medium isusually a suspension of various categories of ingredients (salts, aminoacids, growth regulators, sugars, buffers) that are required for growthof most cell types. However, each specific cell type requires a specificrange of ingredient proportions for growth, and an even more specificrange of formulas for optimum growth. Rate of cell growth will also varyamong cultures initiated with the array of media that permit growth ofthat cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bactoagar, Hazelton agar,Gelrite, and Gelgro are specific types of solid support that aresuitable for growth of plant cells in tissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, maize cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturesrequires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid. Table 1 illustrates the composition of various media useful forcreation of recipient cells and for plant regeneration.

B. Culturing Cells to be Recipients for Transformation

It is believed by the inventors that the ability to prepare andcryopreserve cultures of maize cells is important to certain aspects ofthe present invention, in that it provides a means for reproducibly andsuccessfully preparing cells for particle-mediated transformation,electroporation, or other methods of DNA introduction. The studiesdescribed below set forth techniques which have been successfullyapplied by the inventors to generate transformable and regenerablecultures of maize cells. A variety of different types of media have beendeveloped by the inventors and employed in carrying out various aspectsof the invention. The following table, Table 1, sets forth thecomposition of the media preferred by the inventors for carrying outthese aspects of the invention.

                  TABLE 1                                                         ______________________________________                                        Illustrative Embodiments of Tissue Culture                                     Media Which are Used for Type II Callus                                       Development, Development of Suspension                                        Cultures and Regeneration of Plant Cells                                      (Specifically Maize Cells)                                                                                      OTHER                                                                           BASAL   COMPONENTS**                       MEDIA NO. MEDIUM SUCROSE pH (Amount/L)                                      ______________________________________                                        7       MS*       2%        6.0  .25 mg thiamine                                    .5 mg BAP                                                                     .5 mg NAA                                                                     Bactoagar                                                                 10 MS 2% 6.0 .25 mg thiamine                                                      1 mg BAP                                                                      1 mg 2,4-D                                                                    400 mg L-proline                                                              Bactoagar                                                                 19 MS 2% 6.0 .25 mg thiamine                                                      .25 mg BAP                                                                    .25 mg NAA                                                                    Bactoagar                                                                 20 MS 3% 6.0 .25 mg                                                               1 mg BAP                                                                      1 mg NAA                                                                      Bactoagar                                                                 52 MS 2% 6.0 .25 mg thiamine                                                      1 mg 2,4-D                                                                    10.sup.-7 M ABA                                                               BACTOAGAR                                                                 101 MS 3% 6.0 MS vitamins                                                         100 mg myo-inositol                                                           Bactoagar                                                                 142 MS 6% 6.0 MS vitamins                                                         5 mg BAP                                                                      0.186 mg NAA                                                                  0.175 mg IAA                                                                  0.403 mg 21P                                                                  Bactoagar                                                                 157 MS 6% 6.0 MS vitamins                                                         myo-inositol                                                                  Bactoagar                                                                 163 MS 3% 6.0 MS vitamins                                                         3.3 mg dicamba                                                                100 mg myo-inositol                                                           Bactoagar                                                                 171 MS 3% 6.0 MS vitamins                                                         .25 mg 2,4-D                                                                  10 mg BAP                                                                     100 mg myo-inositol                                                           Bactoagar                                                                 173 MS 6% 6.0 MS vitamins                                                         5 mg BAP                                                                      .186 mg NAA                                                                   .175 mg IAA                                                                   .403 mg 2IP                                                                   10.sup.-7M ABA                                                                200 mg myo-inositol                                                           Bactoagar                                                                 177 MS 3% 6.0 MS vitamins                                                         .25 mg 2,4-D                                                                  10 mg BAP                                                                     10.sup.-7M ABA                                                                100 mg myo-inositol                                                           Bactoagar                                                                 185 MS -- 5.8 3 mg BAP                                                            .04 mg NAA                                                                    RT vitamins                                                                   1.65 mg thiamine                                                              1.38 g L-proline                                                              20 g sorbitol                                                                 Bactoagar                                                                 189 MS -- 5.8 3 mg BAP                                                            .04 mg NAA                                                                    .5 mg niacin                                                                  800 mg L-asparagine                                                           100 mg casaminio                                                              acids                                                                         20 g sorbitol                                                                 1.4 g L-proline                                                               100 mg myo-inositol                                                           Gelgro                                                                    201 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                1 mg 2,4-D                                                                    100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               Gelgro                                                                    205 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                .5 mg 2,4-D                                                                   100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               Gelgro                                                                    209 N6 6% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                100 mg casein                                                                 hydrolysate                                                                   0.69 g L-proline                                                              Bactoagar                                                                 210 N6 3% 5.5 N6 vitamins                                                         2 mg 2,4-D                                                                    250 mg Ca                                                                     pantothenate                                                                  100 mg myo-inositol                                                           790 mg L-asparagine                                                           100 mg casein                                                                 hydrolpate                                                                    1.4 g L-proline                                                               Hazelton agar****                                                             2 mg L-glycine                                                            212 N6 3% 5.5 N6 vitamins                                                         2 mg L-glycine                                                                2 mg 2,4-D                                                                    250 mg Ca                                                                     pantothenate                                                                  100 mg myo-inositol                                                           100 mg casein                                                                 hydrolysate                                                                   1.4 g L-proline                                                               Hazelton agar****                                                         227 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                13.2 mg dicamba                                                               100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               Gelgro                                                                    273 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                1 mg 2,4-D                                                                    16.9 mg AgNO.sub.3                                                            100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                           279 N6 2% 5.8 3.3 mg dicamba                                                      1 mg thiamine                                                                 .5 mg niacin                                                                  800 mg L-asparagine                                                           100 mg casein                                                                 hydrolysate                                                                   100 mg myoinositol                                                            1.4 g L-proline                                                               Gelgro****                                                                288 N6 3%  3.3 mg dicamba                                                         1 mg thiamine                                                                 .5 mg niacin                                                                  .8 g L-asparagine                                                             100 mg myo-inosital                                                           1.4 g L-proline                                                               100 mg casein                                                                 hydrolysate                                                                   16.9 mg AgNO.sub.3                                                            Gelgro                                                                    401 MS 3% 6.0 3.73 mg Na.sub.2 EDTA                                               .25 mg thiamine                                                               1 mg 2,4-D                                                                    2 mg NAA                                                                      200 mg casein                                                                 hydrolysate                                                                   500 mg K.sub.2 SO.sub.4                                                       400 mg KH.sub.2 PO.sub.4                                                      100 mg myo-inositol                                                       402 MS 3% 6.0 3.73 mg Na.sub.2 EDTA                                               .25 mg thiamine                                                               1 mg 2,4-D                                                                    200 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               500 mg K.sub.2 SO.sub.4                                                       400 mg KH.sub.2 PO.sub.4                                                      100 mg myo-inositol                                                       409 MS 3% 6.0 3.73 mg Na.sub.2 EDTA                                               .25 mg thiamine                                                               9.9 mg dicamba                                                                200 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               500 mg K.sub.2 SO.sub.4                                                       400 mg KH.sub.2 PO.sub.4                                                      100 mg myo-inositol                                                       401 Clark's 2% 5.7                                                             Medium***                                                                    607 1/2 × MS 3% 5.8 1 mg thiamine                                           1 mg niacin                                                                   Gelrite                                                                   615 MS 3% 6.0 MS vitamins                                                         6 mg BAP                                                                      100 mg myo-inositol                                                           Bactoagar                                                                 617 1/2 × MS   1.5% 6.0 MS vitamins                                         50 mg myo-inositol                                                            Bactoagar                                                                 708 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                1.5 mg 2,4-D                                                                  200 mg casein                                                                 hydrolysate                                                                   0.69 g L-proline                                                              Gelrite                                                                   721 N6 2% 5.8 3.3 mg dicamba                                                      1 mg thiamine                                                                 .5 mg niacin                                                                  800 mg L-asparagine                                                           100 mg myo-inositol                                                           100 mg casein                                                                 hydrolysate                                                                   1.4 g L-proline                                                               54.65 g mannitol                                                              Gelgro                                                                    726 N6 3% 5.8 3.3 mg dicamba                                                      .5 mg niacin                                                                  1 mg thiamine                                                                 800 mg L-asparagine                                                           100 mg myo-inositol                                                           100 mg casein                                                                 hydrolysate                                                                   1.4 g L-proline                                                           727 N6 3% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                9.9 mg dicamba                                                                100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               Gelgro                                                                    728 N6 3% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                9.9 mg dicamba                                                                16.9 mg AgNO.sub.3                                                            100 mg casein                                                                 hydrolysate                                                                   2.9 g L-proline                                                               Gelgro                                                                    734 N6 2% 5.8 N6 vitamins                                                         2 mg L-glycine                                                                1.5 mg 2,4-D                                                                  14 g Fe sequestreene                                                          (replaces Fe-EDTA)                                                            200 mg casein                                                                 hydrolyste                                                                    0.69 g L-proline                                                              Gelrite                                                                   735 N6 2% 5.8 1 mg 2,4-D                                                          .5 mg niacin                                                                  .91 g L-asparagine                                                            100 mg myo-inositol                                                           1 mg thiamine                                                                 .5 g MES                                                                      .75 g MgCl.sub.                                                               100 mg casein                                                                 hydrolysate                                                                   0.69 g L-proline                                                              Gelgro                                                                    2004 N6 3% 5.8 1 mg thiamine                                                      0.5 mg niacin                                                                 3.3 mg dicamba                                                                17 mg AgNO.sub.3                                                              1.4 g L-proline                                                               0.8 g L-asparagine                                                            100 mg casein                                                                 hydrolysate                                                                   100 mg myo-inositol                                                           Gelrite                                                                   2008 N6 3% 5.8 1 mg thiamine                                                      0.5 mg niacin                                                                 3.3 mg dicamba                                                                1.4 g L-proline                                                               0.8 g L-asparagine                                                            Gelrite                                                                 ______________________________________                                         *Basic MS medium described in Murashige and Skoog (1962). This medium is      typically modified by decreasing the NH.sub.4 NO.sub.3 from 1.64 g/l to       1.55 g/l, and omitting the pyridoxine HCl, nicotinic acid, myoinositol an     glycine.                                                                      **NAA = Napthol Acetic Acid                                                   IAA = Indole Acetic Acid                                                      2IP = 2, isopentyl adenine                                                    2,4D = 2, 4Dichlorophenoxyacetic Acid                                         BAP = 6benzyl aminopurine                                                     ABA = abscisic acid                                                           ***Basic medium described in Clark (1982)                                     ****These media may be made with or without solidifying agent.           

A number of transformable maize cultures have been developed using theprotocols outlined in the following examples. A compilation of thecultures initiated and tested for transformability is set forth in Table2, with the results of the studies given in the two right-hand columns.The Table indicates the general selection protocol that was used foreach of these cultures. The numeral design ations under "Protocol"represent the following:

1. Tissue (suspension) was plated on filters, bombarded and then filterswere transferred to culture medium. After 2-7 days, the filters weretransferred to selective medium. Approximately 3 weeks afterbombardment, tissue was picked from filters as separate callus clumpsonto fresh selective medium.

2. As in 1. above, except after b ombardment the suspension was put backinto liquid--subjected to liquid selection for 7-14 days and thenpipetted at a low density on to fresh selection plates.

3. Callus was bombarded while sitting directly on medium or on filters.Cells were transferred to selective medium 1-14 days after particlebombardment. Tissue was transferred on filters 1-3 times at 2 weeksintervals to fresh selective medium. Callus was then briefly put intoliquid to disperse the tissue onto selective plates at a low density.

4. Callus tissue was transferred onto selective plates one to seven daysafter DNA introduction. Tissue was subcultured as small units of calluson selective plates until transformants were identified.

The totals demonstrate that 27 of 37 maize cultures were transformable.Of those cell lines tested 11 out of 20 have produced fertile plants and7 are in progress. As this table indicates, transformable cultures havebeen produced from ten different genotypes of maize, including bothhybrid and inbred varieties. These techniques for development oftransformable cultures are also important in direct transformation ofintact tissues, such as immature embryos as these techniques rely on theability to select tranformants in cultured cell systems.

                  TABLE 2                                                         ______________________________________                                                                              Fertile                                   Genotype Culture Method Transformable Plants                                ______________________________________                                        A188 × B73                                                                       G(1 × 6)92                                                                         1        +        -                                          G(1 × 6)716 1,2 + +                                                     G(1 × 6)82 1 + +                                                        G(1 × 6)98 1 - NA                                                       G(1 × 6)99 1 - NA                                                       D(1 × 6)122#3 2 - NA                                                    D(1 × 6)114 2 - NA                                                      D(1 × 6)17#33 2 In progress In                                             progress                                                                   HB13-3 3 + In                                                                    progress                                                                   HA133-227 2 - NA                                                              G(6 × 1)17#25 3 + In                                                    C   progress                                                                  ABT4 4 + +                                                                    ABT3 4 + +                                                                    AB60 4 + +                                                                    AB61 4 + +                                                                    AB63 4 + +                                                                    AB80 4 + +                                                                    AB82 4 + In                                                                      progress                                                                   ABT6 4 + ND                                                                   AB12 4 + +                                                                    PH2 4 + +                                                                     AB69 4 + -                                                                    AB44 4 + -                                                                    AB62 4 + ND                                                                  A188 × B84 G(1 × M)82 1 + -                                       A188 × H99 HJ11-7 3 + In                                                    progress                                                                  B73 × A188 G(6 × 1)12#7 2 - NA                                     D(6 × 1)11#43 2 - NA                                                    E1 2 + -                                                                     Hi-II G(CW)31#24  + In                                                            progress                                                                  B73 (6)91#3 2 - NA                                                             (6)91#2 2 - NA                                                               B73- AT824 1,2,3 + +                                                          derived                                                                       N1017A AZ11137a 2 + In                                                            progress                                                                  Cat 100 CB 2 + ND                                                              CC 2 + ND                                                                    A188 E4 2 + -                                                               ______________________________________                                         The symbol indicates that the line was not transformable after 3 attempts     or plants were sterile                                                        NA indicates Not Applicable                                                   ND indicates Not Done                                                    

NA indicates Not Applicable

ND indicates Not Done

EXAMPLE 1 Initiation of the Suspension Culture G(A188×B73)716(designated SC716) for Use in Transformation

This Example describes the development of a maize suspension culture,designated SC716, which was employed in various of the transformationstudies described hereinbelow. The Type II tissue used to initiate thecell suspension was initiated from immature embryos of A188×B73 platedonto N6-based medium with 1 mg/ml 2,4-D (201; see Table 1). A Type IIcallus was initiated by visual selection of fast growing, friableembryogenic cells. The suspension was initiated within 6 months aftercallus initiation. Tissue chosen from the callus to initiate thesuspension consisted of undifferentiated Type II callus. Thecharacteristics of this undifferentiated tissue include the earlieststages of embryo development and soft, friable, undifferentiated tissueunderlying it.

Approximately one gram of tissue was added to 20 mls of liquid medium.In this example, the liquid medium was medium 402 to which differentslow-release growth regulator capsule treatments were added (Adams, W.R., Adams, T. R., Wilston, H. M., Krueger, R. W., and Kausch, A. P,Silicone Capsules for Controlled Auxin Release, in preparation). Thesecapsule treatments included 2,4-D, NAA, 2,4-D plus NAA, and two NAAcapsules. One flask was initiated for each of the different 402 mediaplus growth regulator combinations. Every 7 days each culture wassubcultured into fresh medium by transferring a small portion of thecellular suspension to a new flask. This involved swirling the originalflask to suspend the cells (which tend to settle to the bottom of theculture vessel), tilting the flask on its side and allowing the densercells and cell aggregates to settle slightly. One ml of packed cells wasthen drawn off from this pool of settled cells together with 4 mls ofconditioned medium and added to a flask containing 20 ml fresh medium. Asterile ten ml, wide tip, pipet was used for this transfer (Falcon7304). Any very large aggregates of cells which would not pass easilythrough the pipet tip were excluded. If a growth regulator capsule waspresent, it was also transferred to the new flask.

After approximately 7 weeks, the loose embryogenic cell aggregates beganto predominate and fragment in each of the cultures, reaching a statereferred to as "dispersed." The treatment which yielded the highestproportion of embryogenic clusters was the 402 medium plus one NAAcapsule. After the cultures became dispersed and were doublingapproximately every two to three days as determined by increase inpacked cell volume, a one ml packed cell volume inoculum from eachculture was transferred into 20 ml 401 medium using a ten ml narrow tippipet (Falcon 7551). These transfers were performed about every 31/2days. An inoculum from the 402 plus 2,4-D plus NAA capsules culture wasalso used to initiate a culture in 409 medium (402 without 2,4-D andincluding 10 mg/l dicamba) either with or without 1 ml coconut water(Gibco 670-8130AG) per 25 ml culture medium.

The most dispersed cultures were cryopreserved after 2 weeks, 2 monthsor 5 months.

The culture grown on 409 with coconut water was thawed eight monthsafter cryopreservation, cultured for two weeks on solid 201 culturemedium using BMS as a feeder layer (Rhodes et al., 1988) and transferredto media 409 without coconut water. The culture was maintained bysubculturing twice weekly in 409 medium by the method described above.

EXAMPLE 2 Initiation of the Suspension Culture (A188×B73)82 (designatedSC82) for Use in Transformation

This Example describes the development of another cell line employed invarious of the transformation studies set forth below, termed SC82. Inthe development of SC82, inoculum for suspension culture initiation wasvisually selected from a Type II callus that was initiated from A188×B73immature embryos plated on a N6-based medium containing 13.2 mg/ldicamba (227, Table 1). The suspension culture was initiated within 3months of initiation of the Type II callus. Small amounts (50-100 mg) ofcallus distinguishable by visual inspection because of its highlyproembryonic morphology, were isolated from more mature or organizedstructures and inoculated into a 50 ml flask containing 5 mls offilter-sterilized conditioned medium from the various G(A188×B73) 716suspension cultures (402 medium with four types of capsule treatmentsand 409 medium).

After one week, this 5 ml culture was sieved through a 710 micron meshand used to inoculate 20 mls of corresponding fresh andfilter-sterilized conditioned medium from the established G(A188×B73)716 cultures in 150 ml flasks. After one week or more of growth, two mlsof packed cells were subcultured to fresh media by the method describedabove. The suspension culture maintained on 409 by this method was thencryopreserved within 3 months. The original cell line, which wasmaintained on 409 (not a reinoculated cryopreserved culture) was used inexperiments 1 and 2 months later which resulted in stable transformationand selection (see Table 6 below). The cryopreserved culture was usedfor experiment 6 (see Table 6 below).

EXAMPLE 3 Initiation and Maintenance of Cell Line AT824

This example describes the initiation and maintenance of cell line AT824which has been used routinely for transformation experiments. Immatureembryos (0.5-1.0 mm) were excised from the B73-derived inbred line ATand cultured on N6 medium with 100 uM silver nitrate, 3.3 mg/L dicamba,3% sucrose and 12 mM proline (2004). Six months after initiation type Icallus was transferred to medium 2008. Two months later type I calluswas transferred to a medium with a lower concentration of sucrose (279).A sector of type II callus was identified 17 months later and wastransferred to 279 medium. This cell line is uniform in nature,unorganized, rapid growing, and embryogenic. This culture is desirablein the context of this invention as it is easily adaptable to culture inliquid or on solid medium.

The first suspension cultures of AT824 were initiated 31 months afterculture initiation. Suspension cultures may be inititiated in a varietyof culture media including media containing 2,4-D as well as dicamba asthe auxin source, e.g., media designated 210, 401, 409, 279. Culturesare maintained by transfer of approximately 2 ml packed cell volume to20 ml fresh culture medium at 3 1/2 day intervals. AT824 can beroutinely transferred between liquid and solid culture media with noeffect on growth or morphology.

Suspension cultures of AT824 were initially cryopreserved 33-37 monthsafter culture initiation. The survival rate of this culture was improvedwhen it was cryopreserved following three months in suspension culture.AT824 suspension cultures have been cryopreserved and reinitiated fromcryopreservation at regular intervals since the initial date offreezing. Repeated cycles of freezing have not affected the growth ortransformability of this culture.

EXAMPLE 4 Initiation and Maintenance of Cell lines ABT3, ABT4, ABT6,AB80, AB82, AB12, AB44, AB60, AB61, AB62, AB63, AB69

Friable, embryogenic maize callus cultures were initiated from hybridimmature embryos produced by pollination of inbred A188 plants(University of Minnesota, Crop Improvement Association) with pollen ofinbred line B73 plants (Iowa State University). Ears were harvested whenthe embryos had reached a length of 1.5 to 2.0 mm. The whole ear wassurface sterilized in 50% v/v commercial bleach (2.63% w/v sodiumhypochlorite) for 20 min. at room temperature. The ears were then washedwith sterile distilled, deionized water. Immature embryos wereaseptically isolated and placed on nutrient agar initiation/maintenancemedia with the root/shoot axis exposed to the medium.Initiation/maintenance medium (hereinafter referred to as medium 734)consisted of N6 basal medium (Chu 1975) with 2% (w/v) sucrose, 1.5 mgper liter 2,4-dichlorophenoxyacetic acid (2,4-D), 6 mM proline, and0.25% Gelrite (Kelco, Inc. San Diego). The pH was adjusted to 5.8 priorto autoclaving. Unless otherwise stated, all tissue culturemanipulations were carried out under sterile conditions.

The immature embryos were incubated at 26° C. in the dark. Cellproliferation from the scutellum of the immature embryos were evaluatedfor friable consistency and the presence of well defined somaticembryos. Tissue with this morphology was transferred to fresh media 10to 14 days after the initial plating of the immature embryos. The tissuewas then subcultured on a routine basis every 14 to 21 days. Sixty toeighty milligram quantities of tissue were removed from pieces of tissuethat had reached a size of approximately one gram and transferred tofresh medium. Subculturing always involved careful visual monitoring tobe sure that only tissue of the correct morphology was maintained. Thepresence of somatic embryos ensured that the cultures would give rise toplants under the proper conditions. The cell cultures named ABT3, ABT4,ABT6, AB80, AB82, AB12, AB44, AB60, AB61, AB62, AB63, AB69 wereinitiated in this manner. The cell lines ABT3, ABT4, and ABT6 wereinitiated from immature embryos of a 5-methyltryptophan resistantderivative of A188×B73.

EXAMPLE 5 Initiation and Maintenance of type II Callus of the GenotypeHi-II

The Hi-II genotype of corn was developed from an A188×B73 cross. Thisgenotype was developed specifically for a high frequency of initiationof type II cultures (100% response rate, Armstrong et al., 1991).Immature embryos (8-12 days post-pollination, 1 to 1.2 mm) were excisedand cultured embryonic axis down on N6 medium containing 1 mg/L 2,4-D,25 mM L-proline (201) or N6 medium containing 1.5 mg/L 2,4-D, 6mmL-proline (734). Type II callus can be initiated either with or withoutthe presence of 100 μM AgNo₃. Cultures initiated in the presence ofAgNo₃ was transferred to medium lacking this compound 14-28 days afterculture initiation. Callus cultures were incubated in the dark at 23-28°C. and transferred to fresh culture medium at 14 day intervals.

Hi-II type II callus is maintained by manual selection of callus at eachtransfer. Alternatively, callus can be resuspended in liquid culturemedium, passed through a 1.9 mm sieve and replated on solid culturemedium at the time of transfer. It is believed that this sequence ofmanipulations is one way to enrich for recipient cell types. Regenerabletype II callus that is suitable for transformation can be routinelydeveloped from the Hi-II genotype and hence new cultures are developedevery 6-9 months. Routine generation of new cultures reduces the periodof time over which each culture is maintained and hence insuresreproducible, highly regenerable, cultures that routinely producefertile plants.

EXAMPLE 6 Initiation of Cell Line E1

An ear of the genotype B73 was pollinated by A188. Immature embryos(1.75-2.00 mm) were excised and cultured on 212 medium (see Table 1).About 4 months after embryo excision, approximately 5 ml PCV type IIcallus was inoculated into 50 ml liquid 210 medium (see Table 1). Thesuspension was maintained by transfer of 5 ml suspension to 50 ml fresh210 medium every 3 1/2 days. This suspension culture was cryopreservedabout 4 months after initiation.

D. Cryopreservation Methods

Cryopreservation is important because it allows one to maintain andpreserve a known transformable cell culture for future use, whileeliminating the cummulative detrimental effects associated with extendedculture periods.

Cell suspensions and callus were cryopreserved using modifications ofmethods previously reported (Finkle, 1985; Withers & King, 1979). Thecryopreservation protocol comprised adding a pre-cooled (0° C.)concentrated cryoprotectant mixture stepwise over a period of one to twohours to pre-cooled (0° C.) cells. The mixture was maintained at 0° C.throughout this period. The volume of added cryoprotectant was equal tothe initial volume of the cell suspension (1:1 addition), and the finalconcentration of cryoprotectant additives was 10% dimethyl sulfoxide,10% polyethylene glycol (6000 MW), 0.23 M proline and 0.23 M glucose.The mixture was allowed to equilibrate at 0° C. for 30 minutes, duringwhich time the cell suspension/cryoprotectant mixture was divided into1.5 ml aliquot (0.5 ml packed cell volume) in 2 ml polyethylenecryo-vials. The tubes were cooled at 0.5° C./minute to -8° C. and heldat this temperature for ice nucleation.

Once extracellular ice formation had been visually confirmed, the tubeswere cooled at 0.5° C./minute from -8° C. to -35° C. They were held atthis temperature for 45 minutes (to insure uniform freeze-induceddehydration throughout the cell clusters). At this point, the cells hadlost the majority of their osmotic volume (i.e. there is little freewater left in the cells), and they could be safely plunged into liquidnitrogen for storage. The paucity of free water remaining in the cellsin conjunction with the rapid cooling rates from -35 to -196° C.prevented large organized ice crystals from forming in the cells. Thecells are stored in liquid nitrogen, which effectively immobilizes thecells and slows metabolic processes to the point where long-term storageshould not be detrimental.

Thawing of the extracellular solution was accomplished by removing thecryo-tube from liquid nitrogen and swirling it in sterile 42° C. waterfor approximately 2 minutes. The tube was removed from the heatimmediately after the last ice crystals had melted to prevent heatingthe tissue. The cell suspension (still in the cryoprotectant mixture)was pipetted onto a filter, resting on a layer of BMS cells (the feederlayer which provided a nurse effect during recovery). Dilution of thecryoprotectant occurred slowly as the solutes diffused away through thefilter and nutrients diffused upward to the recovering cells. Oncesubsequent growth of the thawed cells was noted, the growing tissue wastransferred to fresh culture medium. The cell clusters were transferredback into liquid suspension medium as soon as sufficient cell mass hadbeen regained (usually within 1 to 2 weeks). After the culture wasreestablished in liquid (within 1 to 2 additional weeks), it was usedfor transformation experiments. When desired, previously cryopreservedcultures may be frozen again for storage.

E. DNA Segments Comprising Exogenous Genes

As mentioned previously, there are several methods to construct the DNAsegments carrying DNA into a host cell that are well known to thoseskilled in the art. The general construct of the vectors used herein areplasmids comprising a promoter, other regulatory regions, structuralgenes, and a 3' end.

Several plasmids encoding a variety of different genes have beenconstructed by the present inventors, the important features of whichare represented below in Table 3. Certain of these plasmids are alsoshown in FIG. 1: pDPG165, FIG. 1(A, C); pDPG208, FIG. 1(B, D); pDPG141,FIG. 1(E); pDPG237, FIG. 1(F); pDPG313 through pDPG319, FIG. 1(H)through FIG. 1(N); pDPG290, FIG. 1(O); pDPG300 through pDPG304, FIG.1(P) through FIG. 1(S); pDPG386 through pDPG389, FIG. 1(T) through FIG.1(W); pDPG140, FIG. 1(X); pDPG172, FIG. 1(Y); pDPG425, FIG. 1(Z);pDPG427, FIG. 1(AA); pDPG451, FIG. 1(BB); pDPG 354, FIG. 1(CC); pDPG344,FIG. 1(DD); pDPG337, FIG. 1(EE).

                  TABLE 3                                                         ______________________________________                                        RECOMBINANT                                                                     VECTOR   DELIBERATE                                                           DESIGNATION & PARENT INSERT EXPRESSION                                        SOURCE REPLICON DNA ATTEMPT                                                 ______________________________________                                        pDPG140       pUC19     1, 118, 102,                                                                            1                                               103                                                                         pDPG141 pUC19 1, 100, 1                                                         101                                                                         pDPG165 pUC19 2, 100, 2                                                         101                                                                         pDPG172 pUC19 3, 118, 102, 3                                                    103                                                                         pDPG182 pUC19 2, 118, 102, 2                                                    103                                                                         pDPG205 pVK101 1, 101, 1                                                        102                                                                         pDPG208 pUC19 1, 100 1                                                        pDPG215 pUC18 3, 100, 3                                                         102, 103                                                                    pDPG215 pUC19 3, 100, 3                                                         101, 102                                                                    pDPG226 pUC19 4, 100, 4                                                         101                                                                         pDPG230--231, 251, 262- pUC19 1, 2, 100, 1, 2                                 264, 279, 282, 283  101                                                       pDPG238-239 pUC19 2, 4, 100, 2, 4                                               101                                                                         pDPG240-241 pUC19 2, 5, 100, 2, 5                                               101                                                                         pDPG243-244 pUC19 2, 6, 100, 2, 6                                               101                                                                         pDPG246 pUC19 7, 100, 7                                                         101                                                                         pDPG265 pBR325 9, 100 9                                                       pDPG266-267 pUC19 8, 100, 8                                                     101                                                                         pDPG268-269 pUC19 1, 100, 1                                                     102                                                                         pDPG270-273 pUC19 1, 100, 1                                                     101                                                                         pDPG274 pUC19 9, 100, 9                                                         101                                                                         pDPG275 pUC18 10, 3, 100 10, 3                                                pDPG287 pUC19 2, 103, 2                                                         105                                                                         pDPG288 pUC13 11, 100, 11                                                       103                                                                         pDPG290 pUC19 12, 100, 12                                                       103                                                                         pDPG291 pUC19 1, 103, 1                                                         104                                                                         pDPG300 pUC19 2, 12, 2, 12                                                      100, 101,                                                                     102, 103                                                                    pDPG301 pUC19 2, 12, 2, 12                                                      100, 101,                                                                     102, 103                                                                    pDPG302 pUC18 2, 12, 2, 12                                                      100, 101,                                                                     102, 103                                                                    pDPG303 pUC18 2, 12, 2, 12                                                      100, 101,                                                                     102, 103                                                                    pDPG304 ColE1 13, 100, 13                                                       103                                                                         pDPG313 pUC18 2, 4, 100, 2, 4                                                   103, 106,                                                                     107                                                                         pDPG314 pUC19 2, 4, 100, 2, 4                                                   103, 106,                                                                     107                                                                         pDPG315 pUC23 2, 4, 100, 2, 4                                                   103, 106,                                                                     108                                                                         pDPG316 pUC23 2, 4, 100, 2, 4                                                   103, 106,                                                                     108                                                                         pDPG317 pUC23 2, 4, 100, 2, 4                                                   103, 106,                                                                     107                                                                         pDPG318 pUC23 2, 4, 100, 2, 4                                                   103, 106,                                                                     109                                                                         pDPG319 pUC23 2, 4, 100, 2, 4                                                   103, 106,                                                                     109                                                                         pDPG320 pUC18 11, 100, 11                                                       102, 103                                                                    pDPG324 pUC19 1, 2, 100, 1, 2                                                   101                                                                         pDPG325 pUC19 1, 2, 100, 1, 2                                                   101                                                                         pDPG326 pUC19 1, 2, 100, 1, 2                                                   101, 102                                                                    pDPG327 pUC19 1, 2, 100, 1, 2                                                   101, 102                                                                    pDPG328 pUC19 1, 2, 100, 1, 2                                                   101, 113                                                                    pDPG329 pUC19 1, 2, 100, 1, 2                                                   101, 113                                                                    pDPG332 pUC19 14, 100, 14                                                       110                                                                         pDPG334 gGEM3 15, 111, 15                                                       121, 103                                                                    pDPG335 pUC119 15, 112, 15                                                      122, 114                                                                    pDPG336 pIC20H 19, 100, 19                                                      117, 103                                                                    pDPG337 plC20H 19, 100, 19                                                      117, 103                                                                    pDPG338 pUC120 16, 112, 16                                                      114, 115                                                                    pDPG339 pUC119 17, 111, 17                                                      103                                                                         pDPG340 pUC19 18, 111, 18                                                       103                                                                         pDPG344 pSK 11, 100, 11                                                         102, 110                                                                    pDPG345 pSK 2, 14, 2, 14                                                        100, 102,                                                                     110, 103                                                                    pDPG346 pSK 2, 14, 2, 14                                                        100, 102,                                                                     110, 103                                                                    pDPG347 pSK 2, 14, 2, 14                                                        100, 102,                                                                     110, 103                                                                    pDPG348 pSK 2, 14, 2, 14                                                        100, 102,                                                                     110, 103                                                                    pDPG351 pUC19 3, 100, 3                                                         101                                                                         pDPG354 pSK- 19, 100, 19                                                        101, 110                                                                    pDPG355 pUC19 1, 100, 1                                                         102, 103                                                                    pDPG356 pUC19 1, 100, 1                                                         116, 103                                                                    pDPG357 plC20H 1, 100, 1                                                        117, 103                                                                    pDPG358 pUC8 1, 118, 1                                                          102, 103                                                                    pDPG359 pUC119 1, 119, 1                                                        103                                                                         pDPG360 pUC119 1, 120, 1                                                        103                                                                         pDPG361 pUC19 1, 111, 1                                                         103                                                                         pDPG362 pUC120 1, 112, 1                                                        103                                                                         pDPG363 pSP73 2, 100, 2                                                         102, 103                                                                    pDPG364 pUC119N 20, 100 20                                                    pDPG365 pUC19 21, 100, 21                                                       103                                                                         pDPG366 pSP73 22, 100, 22                                                       102, 103                                                                    pDPG367 pBS+ 22, 100, 22                                                        102, 103                                                                    pDPG368 pSP73 24, 100, 24                                                       102, 103                                                                    pDPG369 plC20H 24, 100, 24                                                      117, 103                                                                    pDPG370 pBS+ 5, 100, 5                                                          116, 103                                                                    pDPG371 pSP73 15, 100, 15                                                       121, 103                                                                    pDPG372 pUC119 15, 100, 15                                                      122, 103                                                                    pDPG373 pUC119 16, 120, 16                                                      115                                                                         pDPG374 pUC120 16, 112, 16                                                      114                                                                         pDPG375 pUC119 16, 111, 16                                                      115                                                                         pDPG376 pUC119 18, 111, 18                                                      103                                                                         pDPG377 pUC119 18, 111, 18                                                      103                                                                         pDPG378 pUC119 17, 111, 17                                                      103                                                                         pDPG379 pUC119 17, 111, 17                                                      103                                                                         pDPG380 pUC119 17, 111, 17                                                      103                                                                         pDPG381 plC20H 19, 100, 19                                                      117, 103                                                                    pDPG382 plC20H 19, 100, 19                                                      117, 103                                                                    pDPG384 pUC8 19, 100, 19                                                        102, 103                                                                    pDPG385 pSP73 23, 100, 23                                                       121, 103                                                                    pDPG386 pACYC177 6, 114 6                                                     pDPG387 pACYC177 1, 6, 100, 11 1, 6                                             4                                                                           pDPG388 pACYC177 2, 6, 100, 10 2, 6                                             3, 114                                                                      pDPG389 pACYC177 2, 6, 100, 10 2, 6                                             3, 114,                                                                     pDPG391 pUC8 19, 100, 19                                                        102, 103                                                                    pDPG392 pUC19 2, 100, 2                                                         101                                                                         pDPG393 pSK- 4, 109, 4                                                          102, 106,                                                                     103                                                                         pDPG394 pSK- 4, 124, 4                                                          106, 103                                                                    pDPG396 pBS+ 19, 100, 19                                                        102, 7,                                                                       103, 121                                                                    pDPG404 pSK- 1, 108, 1                                                          103                                                                         pDPG405 pSK- 1, 107, 1                                                          103                                                                         pDPG406 pSK- 1, 109, 1                                                          103                                                                         pDPG407 pSK- 1, 109, 1                                                          102, 103                                                                    pDPC408 pSK- 1, 124, 1                                                          103                                                                         pDPG411 pBS+ 19, 100, 19                                                        103, 121                                                                    pDPG415 pUC8 19, 118, 19                                                        103                                                                         pDPG418 pGEM 15, 122, 15                                                        123                                                                         pDPG419 pUC19 3, 100, 101, 3                                                    116                                                                         pDPG420 pUC9 3, 100, 101, 3                                                     116                                                                         pDPG421 pSP72 2, 103, 126 2                                                   pDPG422 pBS 1, 103, 126 1                                                     pDPG424 pSK- 4, 124, 4                                                          102, 103                                                                    pDPG427 pSK- 25, 124, 25                                                        102, 103                                                                    pDPG434 pSK- 25, 103, 126 25                                                  pDPG436 pSK- 25, 100, 102, 25                                                   103                                                                         pDPG437 pUC19 2.100, 101, 2                                                     102                                                                         pDPG438 pUC19 2, 100, 101, 2                                                    102                                                                         pDPG439 pUC19 2, 100, 101, 2                                                    102                                                                         pDPG441 pSK- 25, 103, 108 25                                                  pDPG443 pSK- 25, 100, 103, 25                                                   108                                                                         pDPG447 pSK- 25, 102, 103, 25                                                   109                                                                         pDPG451 pUC18 26, 100, 102, 26                                                  103                                                                         pDPG452 pGEM 2, 123 2                                                         pDPG453 pUC19 2, 103, 127 2                                                   pDPG456 pSK- 19, 100, 19                                                        101, 110                                                                    pDPG457 pSK- 19, 100, 19                                                        101, 110                                                                    pDPG458 pSK- 1, 103, 127 1                                                    pDPG465 pSK- 25, 100, 101, 25                                                   102                                                                         pDPG467 pSK- 25, 102, 118 25                                                  pDPG469 pUC19 26, 118, 102, 26                                                  103                                                                         pDPG474 pUC19 27, 100, 102, 27                                                  103                                                                       ______________________________________                                    

KEY Insert DNA and Deliberate Expression Attempt

1. The uidA gene from E. Coli encodes β-glucuronidase (GUS). Cellsexpressing uidA produce a blue color when given the appropriatesubstrate. Jefferson, R. A. 1987. Plant Mol. Biol. Rep 5: 387-405.

2. The bar gene from Streptomyces hygroscopicus encodes phosphinothricinacetyltransferase (PAT). Cells expressing PAT are resistant to theherbicide Basta. White, J., Chang, S.-Y. P., Bibb, M. J., and Bibb, M.J. 1990. Nucl. Ac. Research 18: 1062.

3. The lux gene from firefly encodes luciferase. Cells expressing luxemit light under appropriate assay conditions. dewet, J. R., Wood, K.V., DeLuca, M., Helinski, D. R., Subramani, S. 1987. Mol. Cell. Biol. 7:725-737.

4. The aroA gene from Salmonella typhimurium encodes5-enolpyruvylshikimate 3-phosphate synthase (EPSPS). Comai, L., Sen, L.C., Stalker, D. M., Science 221: 370-371, 1983.

5. The dhfr gene from mouse encodes dihydrofolate reductase (DHFR).Cells expressing dhfr are resistant to methotrexate. Eichholtz, D. A.,Rogers, S. G., Horsch, R. B., Klee, H. J., Hayford, M., Hoffman, N. L.,Bradford, S. B., Fink, C., Flick, J., O'Connell, K. M., Frayley, R. T.1987. Somatic Cell Mol. Genet. 13: 67-76.

6. The neo gene from E. Coli encodes aminoglycoside phosphotransferase(APH). Cells expressing neo are resistant to the aminoglycosideantibiotics. Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B.,Schaller, H. 1982. Gene 19: 327-336.

7. The amp gene from E. Coli encodes β-lactamase. Cells expressingβ-lactamase produce a chromogenic compound when given the appropriatesubstrate. Sutcliffe, J. G. 1978. Proc. Nat. Acad. Sci. USA 75:3737-3741.

8. The xylE gene from Ps. putida encodes catechol dihydroxygenase. Cellsexpressing xylE produce a chromogenic compound when given theappropriate substrate. Zukowsky et al. 1983. Proc. Nat. Acad. Sci. USA80: 1101-1105.

9. The R,C1 and B genes from maize encode proteins that regulateanthocyanin biosynthesis in maize. Goff, S., Klein, T., Ruth, B., Fromm,M., Cone, K., Radicella, J., Chandler, V. 1990. EMBO J.: 2517-2522.

10. The als gene from Zea mays encodes acetolactate synthase. The enzymewas mutated to confer-resistance to sulfonylurea herbicides. Cellsexpressing als are resistant to the herbicide Gleen. Yang, L. Y., Gross,P. R., Chen, C. H., Lissis, M. 1992. Plant Molecular Biology 18:1185-1187.

11. The proteinase inhibitor II gene was cloned from potato and tomato.Plants expressing the proteinase inhibitor II gene show increasedresistance to insects. Potato: Graham, J. S., Hall, G., Pearce, G.,Ryan, C. A. 1986 Mol. Cell. Biol. 2: 1044-1051. Tomato: Pearce, G.,Strydom, D., Johnson, S., Ryan, C. A. 1991. Science 253: 895-898.

12. The Bt gene from Bacillus thuringensis berliner 1715 encodes aprotein that is toxic to insects. Plants expressing this gene areresistant to insects. This gene is the coding sequence of Bt 884modified in two regions for improved expression in plants. Vaeck, M.,Reynaerts, A., Hofte, H., Jansens, S., DeBeuckeleer, M., Dean, C.,Aeabeau, M., Van Montagu, M., and Leemans, J. 1987. Nature 328: 33-37.

13. The bxn gene from Klebsiella ozaeneae encodes a nitrilase enzymespecific for the herbicide bromoxynil. Cells expressing this gene areresistant to the herbicide bromoxynil. Stalker, D. m., McBride, K. E.,and Malyj, L. Science 242: 419-422, 1988.

14. The WGA-A gene encodes wheat germ agglutinin. Expression of theWGA-A gene confers resistance to insects. The WGA-A gene was cloned fromwheat. Smith, J. J., Raikhel, N. V. 1989. Plant Mol. Biology 13:601-603.

15. The dapA gene was cloned from E. Coli. The dapA gene codes fordihydrodipicolinate synthase. Expression of this gene in plant cellsproduces increased levels of free lysine. Richaud, F., Richaud, C.,Rafet, P. and Patte, J. C. 1986. J. Bacteriol. 166: 297-300.

6. The Z10 gene codes for a 10 kd zein storage protein from maize.Expression of this gene in cells alters the quantities of 10 kD Zein inthe cells. Kirihara, J. A., Hunsperger, J. P., Mahoney, W. C., andMessing, J. 1988. Mol. Gen. Genef. 211: 477-484.

17. The A20 sequence encodes the 19 kd zein storage protein of Zea mays.Expression of the construct in maize alters quantities of the native 19kd zein gene.

18. The Z4 sequence is for the 22 kd zein storage protein of Zea mays.Expression of this construct in maize alters quantities of the native 22kd zein gene.

19. The Bt gene cloned from Bacillus thuringensis Kurstaki encodes aprotein that is toxic to insects. The gene is the coding sequence of thecry IA(c) gene modified for improved expression in plants. Plantsexpressing this gene are resistant to insects. Hofte, H. and Whiteley,H. R., 1989. Microbiological Reviews. 53: 242-255.

20. The als gene from Arabidopsis thaliana encodes a sulfonylureaherbicide resistant acetolactate synthase enzyme. Cells expresing thisgene are resistant to the herbicide Gleen. Haughn, G. W., Smith, J.,Mazur, B., and Somerville, C. 1988. Mol. Gen. Genet. 211: 266-271.

21. The deh1 gene from Pseudomonas putida encodes a dehalogenase enzyme.Cells expressing this gene are resistant to the herbicide Dalapon.Buchanan-Wollaston, V., Snape, A., and Cannon, F. 1992. Plant CellReports 11: 627-631.

22. The hygromycin phosphotransferase II gene was isolated from E. coli.Expression of this gene in cells produces resistance to the antibiotichygromycin.

Waldron, C., Murphy, E. B., Roberts, J. L., Gustafson, G. D., Armour, S.L., and Malcolm, S. K. Plant Molecular Biology 5: 103-108, 1985.

23. The lysC gene from E. coli encodes the enzyme aspartyl kinase III.Expression of this gene leads to increased levels of lysine in cells.

24. The hygromycin phosphotransferase II gene was isolated fromStreptomyces hygroscopicus. Expression of this gene in cells producesresistance to the antibiotic hygromycin.

25. The EPSPS gene (5-enolpyruvy/shikimate -3-phosphate synthase) genefrom Zea Mays was mutated to confer resistance to the herbicideRoundup®. An isoleucine has been substituted for threonine at amino acidposition 102 and a serine has been substituted for proline at amino acidposition 106.

26. The mtlD gene was cloned from E. coli. This gene encodes the enzymemannitol-1-phosphate dehydrogenase. Lee and Saier, 1983. J. ofBacteriol. 153:685.

27. The HVA-1 gene encodes a Late Embryogenisis Abundant (LEA) protein.This gene was isolated from barley. Dure, L., Crouch, M., Harada, J.,Ho, T.-H. D. Mundy, J., Quatrano, R, Thomas, T, and Sung, R., PlantMolecular Biology 12: 475-486.

Regulatory Sequences

100. Promoter sequences from the Cauliflower Mosaic Virus genome. Odell,J. T., Nagy, F., and Chua, N.-H. 1985. Nature 313: 810-812.

101. Promoter and terminator sequences from the Ti plasmid ofAgrobacterium tumefaceiens. (a) Bevan, M., 1984. Nucleic Acid Research12: 8711-8721; (b) Ingelbrecht, I. L. W., Herman, L. M. F., DeKeyser, R.A., Van Montagu, M. C., Depicker, A. G. 1989. The Plant Cell 1: 671-680;(c) Bevan, M., Barnes, W. M., Chilton, M. D., 1983. Nucleic AcidResearch. 11: 369-385; (d) Ellis, J. G., Llewellyn, D. J., Walker, J.C., Dennis, E. S., Peacocu, W. J. 1987. EMBO J. 6: 3203-3208.

102. Enhancer sequences from the maize alcohol dehydrogenase gene.Callis, J., Fromm, M. E., Walbot, V., 1987. Genes Dev. 1: 1183-1200.

103. Terminator sequences from Ti plasmid of Agrobacterium tumefaciens.(a) Bevan, M., 1984. Nucleic Acid Research 12: 8711-8721; (b)Ingelbrecht, I. L. W., Herman, L. M. F., DeKeyser, R. A., Van Montagu,M. C., Depicker, A. G. 1989. The Plant Cell 1: 671-680; (c) Bevan, M.,Barnes, W. M., Chilton, M. D., 1983. Nucleic Acid Research. 11: 369-385.

104. Pollen specific promoter sequence ZM13 from maize. 105. Transitpeptide sequence from rbcS gene from pea. Fluhr, R., Moses, P., Morelli,G., Coruzzi, C., Chua, N.-H. 1986. EMBO J. 5: 2063-2071.

106. Optimized transit peptide sequence consisting of sequences fromsunflower and maize. Constructed by Rhone Poulenc Agrochimie.

107. Fused promoter sequences from Cauliflower Mosaic Virus genome andArabidopsis thaliana H4 histone gene. Constructed by Rhone PoulencAgrochimie.

108. Promoter sequence from Arabidopsis thaliana histone H4 gene.Chaboute, H. E., Chambet, N., Philipps, G., Ehling, M. and Grigot, C.1987. Plant Mol. Biol. 8: 179-191.

109. Promoter sequence from maize α-tubulin gene.

110. Terminator sequences from the potato proteinase inhibitor II gene.An, G., Mitra, A., Choi, H. K., Costa, M. A., An, K., Thornburg, R. W.,Ryan, C. A. 1989. Plant Cell 1: 115-122.

111. Promoter from the maize 10 kd zein gene.

112. Promoter from the maize 27 kd zein gene. Ueda, T. and Messing, J.1991. Theor. Appl. Genet. 82: 93-100.

113. The matrix attachment region (MAR) was isolated from chicken. Useof this DNA sequence reduces variations in gene expression due tointegration position effects. Stief, A., Winter, D., Stratling, W. H.,Sippel, A. E. 1989. Nature 341: 343.

114. Terminator sequences from the Cauliflower Mosaic Virus genome.Timmermans, M. C. P., Maliga, P., Maliga, P., Vieiera, J. and Messing,J. 1990. J. Biotechnol. 14: 333-344.

115. Terminator from maize 10 kd zein gene. Kirihara, J. A., Hunsperger,J. P., Mahoney, W. C., Messing, J. 1988. Mol. Gen. Genet. 211: 477-484.

116. Enhancer sequence from the shrunken-1 gene of Zea mays. Vasil, V.,Clancy, M., Ferl, R. J., Vasil, I. K., Hannah, L. C. 1989. PlantPhysiol. 91: 1575-1579.

117. RNA leader sequence from the ribulose-bis-phosphate carboxylasegene from Glycine max. Joshi, C. P. 1987. Nucleic Acids Res.15:6643-9640.

118. Promoter sequence from the alcohol dehydrogenase gene of Zea mays.Walker, J. C., Howard, E. A., Dennis, E. S., Peacock, W. J. 1987.P.N.A.S. 84: 6624-6628.

119. Promoter sequence from the glutamine synthetase gene of Zea mays.

120. Promoter sequence from the 22 kD (Z4) zein gene of Zea mays.Schmidt, R. J., Ketudat, M., Ankerman, M. J. and Hoschek, G. 1992. PlantCell 4: 689-700.

121. Transit peptide sequence of the ribulose bis-phosphate carboxylasesmall subunit gene from Zea mays. GenBank Accession Y00322.

122. Transit peptide sequence of the dihydropicolinic acid synthase geneof Zea mays.

123. Globulin-1, glb1, promoter and terminator sequences from Zea mays.Belanger and Kriz. 1991

124. Promoter sequence from maize histone H3C4. Chaubet, N., Clement,B., Philipps, G. and Gigot, C. 1991. Plant Molecular Biology 17:935-940.

125. DNA sequence encoding first 8 amino acids of the mature ribulosebisphosphate carboxylase gene of Zea mays.

126. Actin-1 5' region including promoter from Zea mays. Wang, Y.,Zhang, W., Cao, J., McEhoy, D. and Ray Wu. 1992. Molecular and CellularBiology 12: 3399-3406.

127. The DS element isolated from Zea mays.

DNA segments encoding the bar gene were constructed into a plasmid,termed pDPG165, which was used to introduce the bialaphos resistancegene into recipient cells (see FIGS. 1A and C). The bar gene was clonedfrom Streptomyces hygroscopicus (White et al., 1990) and exists as a559-bp Sma I fragment in plasmid pIJ4101. The sequence of the codingregion of this gene is identical to that published (Thompson et al.,1987). To create plasmid pDPG165, the Sma I fragment from pIJ4104 wasligated into a pUC19-based vector containing the Cauliflower MosaicVirus (CaMV) 35S promoter (derived from pBI221.1. provided by R.Jefferson, Plant Breeding Institute, Cambridge, England), a polylinker,and the transcript 7 (Tr7) 3' end from Agrobacterium tumefaciens (3' endprovided by D. Stalker, Calgene, Inc., Davis, Calif.).

An additional vector encoding GUS, pDPG208, (FIGS. 1B and 1D) was usedin these experiments. It was constructed using a 2.1 kb BamHI/EcoRIfragment from pAGUS1 (provided by J. Skuzeski, University of Utah, SaltLake City, Utah) containing the coding sequence for GUS and the nos3'-end from Agrobacterium tumefaceiens. In pAGUS1 the 5'-noncoding and5'-coding sequences for GUS were modified to incorporate the Kozakconsensus sequence (Kozak, 1984) and to introduce a new HindIIIrestriction site 6 bp into the coding region of the gene (see FIG. 1E).The 2.1 kb BamHI/EcoRI fragment from pAGUS1 was ligated into a 3.6 kbBamHI/EcoRI fragment of a pUC19-based vector pCEV1 (provided by Calgene,Inc., Davis, Calif.). The 3.6 kb fragment from pCEV1 contains pUC19 anda 430 bp 35S promoter from cauliflower mosaic virus adjacent to thefirst intron from maize Adh1.

In terms of a member of the R gene complex for use in connection withthe present invention, the most preferred vectors contain the 35Spromoter from Cauliflower Mosaic Virus, the first intron from maize Adh1gene, the Kozak consensus sequence, Sn:bol3 cDNA, and the transcript 73' end from Agrobacterium tumefaceiens. One such vector prepared by theinventors is termed pDPG237. To prepare pDPG237 (see FIG. 1F), the cDNAclone of Sn:bol3 was obtained from S. Dellaporta (Yale University, USA).A genomic clone of Sn was isolated from genomic DNA of Sn:bol3 which hadbeen digested to completion with HindIII, ligated to lambda arms andpackaged in vitro. Plaques hybridizing to two regions of cloned Ralleles, R-nj and R-sc (Dellaporta et al., 1988) were analyzed byrestriction digest. A 2 kb Sst-HincII fragment from the pSn7.0 was usedto screen a cDNA library established in lambda from RNA oflight-irradiated scutellar nodes of Sn:bol3. The sequence and arestriction map of the cDNA clone was established.

The cDNA clone was inserted into the same plant expression vectordescribed for pDPG165, the bar expression vector (see above), andcontains the 35S Cauliflower mosaic virus promoter, a polylinker and thetranscript 7 3' end from Agrobacterium tumefaceiens. This plasmid,pDPG232, was made by inserting the cDNA clone into the polylinkerregion; a restriction map of pDPG232 is shown in FIG. 1G. The preferredvector, pDPG237, was made by removing the cDNA clone and Tr7 3' end frompDPG232, with AvaI and EcoRI and ligating it with a BamHI/EcoRI fragmentfrom pDPG208. The ligation was done in the presence of a BamHI linker asfollows (upper strand, seq id no:3; lower strand, seq id no:4):

GATCCGTCGACCATGGCGCTTCAAGCTTC

GCAGCTGGTACCGCGAAGTTCGAAGGGCT

The final construct of pDPG237 contained a Cauliflower mosaic virus 35Spromoter, the first intron of Adh1, Kozak consensus sequence, the BamHIlinker, cDNA of Sn:Bol3, and the Tr7 3' end and is shown in FIG. 1F.

Additional vectors have been prepared using standard genetic engineeringtechniques. For example, a vector, designated pDPG128, has beenconstructed to include the neo coding sequence (neomycinphosphotransferase (APH(3')-II)). Plasmid pDPG128 contains the 35Spromoter from CaMV, the neomycin phosphotransferase gene from Tn5 (Berget al., 1980) and the Tr7 terminator from Agrobacterium tumefaceiens.Another vector, pDPG154, incorporates the crystal toxin gene and wasalso prepared by standard techniques. Plasmid pDPG154 contains the 35Spromoter, the entire coding region of the crystal toxin protein ofBacillus thuringiensis var. kurstaki HD 263, and the Tr7 promoter.

Various tandem vectors have also been prepared. For example, a bar/aroAtandem vector (pDPG238) was constructed by ligating a blunt-ended 3.2 kbDNA fragment containing a mutant EPSP synthase aroA expression unit(Barkai-Golan et al., 1978) to NdeI-cut pDPG165 that had been bluntedand dephosphorylated (NdeI introduces a unique restriction cutapproximately 200 bp downstream of the Tr7 3'-end of the bar expressionunit). Transformants having aroA in both orientations relative to barwere identified.

Additional bar gene vectors employed are pDPG284 and pDPG313-pDPG319(FIG. 1H-N), the latter series being obtained from Rhone-PoulencAgrochimie (RPA). The orientation of the bar gene in DPG165 was invertedwith respect to the pUC vector, to obtain pDPG284. An additional 32 bpof DNA has been inserted into the NdeI site of pDPG165 and pDPG284, toobtain pDPG295 and pDPG297 and pDPG298, respectively. This extra 32 bpof sequence preserves the unique NdeI site in each vector and adds fouradditional restriction sites with the following orientation, relative tothe unique NarI site:

NarI-52 bp-HpaI-NotI-NruI-EcoRI-NdeI.

Tandem bar-aroA vectors with a 35S-histone promoter (constitutive andmeristem enhanced expression) in convergent (pDPG314), divergent(pDPG313), and colinear orientations (pDPG317) have also been employed,as have aroA vectors with a histone promoter (meristem-specificpromoter, (pDPG315/pDPG316) and an α-tubulin promoter (root-specific,pDPG318/pDPG319) in colinear and divergent orientations to barrespectively.

Plasmid pDPG290 incorporates the modified Bt CryIA(b) gene, IAb6,obtained from Plant Genetic Systems, PGS. A 1.8 kb NcoI-NheI fragmentcontaining IAb6 was ligated together with the Tr7 3' region on a 0.5 KbNheI-EcoRI fragment from pDPG145 and 3.6 Kb NcoI-EcoRI fragment frompDGP208 containing the 35S promoter, Adh1 intron I, and the requiredplasmid functions to create pDPG290 (FIG. 1, O).

In the construction of IAb6-bar tandem vectors, modifications were madeto the separate bar and IAb6 expression units so that IAb6 could beinserted as a NotI-flanked cassette into the bar-containing plasmid. ANotI recognition site was introduced through a series of ligations intoplasmids which contained the bar expression unit in two orientationswith respect to the new NotI recognition site. The bar expression unitwas removed from pDPG165 as a HindIII-EcoRI fragment and ligated toHindIII-EcoRI cut pUC18 to give the plasmid pDPG284. This reversed theorientation of the bar expression unit with respect to the pUC plasmid.An oligonucleotide containing HpaI, PmiI, NruI, EcoRI, and NdeIrestriction sites was ligated to NdeI cut pDPG165 and pDPG284 to producepDPG285/pDPG286 and pDPG292/pDPG293, respectively. These pairs ofplasmids were the two orientations of the oligonucleotide. These fourplasmids were then cut with Pmll in order to introduce a NotI linkerwhich destroyed the Pmll recognition site. Plasmids pDPG292 and pDPG293containing the NotI linker were designated pDPG298 and pDPG297,respectively. A NotI site was introduced adjacent to the 3' end of theTr7 3' region by removing a 200 bp EcoRI fragment from pDPG294. Thisvector was designated pDPG296.

An IAb6 vector with flanking NotI sites was constructed via a 4-wayligation consisting of the Tr7 3' region (as a 500 bp HpaI-BglIIfragment from pDPG296), the IAb6 gene (as a 1.8 Kb BamHI-NcoI fragmentfrom pDPG290), the 35S promoter-Adh1 intron 1 cassette (as a 800 bpXbaI-NcoI fragment from pDPG208), and the pBluescript II SK(-)plasmid(as a 2.95 Kb XbaI-HindII fragment). The pBluescript plasmid has apolylinker that positioned a second NotI recognition site next to the 5'end of the IAb6 expression unit. The plasmid which contained theNotI-flanked IAb6 expression unit was designated pDPG299. The IAb6expression unit was excised from pDPG299 as a 3.1 kb NotI fragment andligated to NotI cut pDPG295 and pDPG298 to yield pDPG300/pDPG301 andpDPG302/pDPG303, respectively (FIG. 1P-S). The pairs are the twoorientations possible for each ligation.

A plasmid DNA, pDPG310, was constructed that contains a bar expressionunit and a single copy of the matrix attachment region from the chickenlysozyme gene. The nuclear matrix attachment region (MAR, or"A-element") from the chicken genomic DNA region 5' to the lysozyme geneis contained on a 2.95 kb KpnI-PstI fragment in plasmid pUC19 B1-X1(received from A. E. Sippel, Freiburg). It has been reported that genesflanked by MAR elements result in tissue independent gene expression andenhanced transcriptional activity. Plasmid DNA pDPG310 was constructedby performing a 3-way litigation among the following DNAs:

1) 2.9 kb NotI-KpnI fragment from pBluescript II SK(-),

2) 2.95 kb KpnI-PstI MAR fragment from pUC19 B1-X1, and

3) 2.1 kp PstI-NotI bar-containing fragment from pDPG294.

Plasmid pDPG310 contains three SacI sites, one in each of the above DNAfragments. A second MAR element was inserted into the SacI site at theend of the pBluescript II SK(-) multiple cloning region. The resultingplasmid has a unique NotI site, into which future traits of interestcould be cloned.

In regard to vectors encoding protease inhibitors, plasmid DNA, pRJ15,containing the genomic DNA sequence for the potato pinII gene, wasobtained from Clarence Ryan (Washington State University) and renamedpDPG288. Another plasmid DNA, pT2-47, containing the cDNA sequence forthe tomato pinII gene, was obtained from Clarence Ryan and renamedpDPG289. The potato pinII gene in pDPG288 is flanked by a 35S promoterand a Tr7 3' end.

Tandem vectors were constructed containing a bar expression unit and apotato or tomato pinII expression unit in either convergent or colinearorientation. In each construct, the bar and pinII expression units werecontained on a HindIII fragment that also contains a 1.9 kb fragment ofAdh1 but lacks the Amp^(R) gene and the plasmid origin of replication.The 1.9 kb Adh1 region provides a locus for recombination into the plantgenome without disruption of either the bar or pinII expression units.These HindIII fragments are available for bombardment experiments aseither linear, circular, or supercoiled DNAs.

To construct a plant expression vector containing the potato pinIIterminator, the potato pinII terminator was cloned into pBluescript IISK(-) via a three-part ligation. To obtain the pinII terminator, a 3.7kb PstI fragment was first removed from pDPG288 and subsequentlydigested with RsaI/PstI to yield the 930 bp pinII terminator. ThepBluescript II vector was digested with ScaI/PstI and ScaI/SmaI in twoseparate reactions; the appropriate two plasmid fragments weregel-purified. These fragments and the pinII terminator were ligated togive plasmid pDPG331.

In order to construct pDPG320, a pinII expression vector containing the35S promoter-AdhI introni-pinII (with intron and terminator)-Tr7terminator vector, the following protocol was used. pDPG309 was cut withBglII/EcoRI and the vector fragment was isolated. pDPG157 was cut withPstI/EcoRI and the 500 bp Tr7 fragment was isolated. To isolate the AdhIintron 3'-end, PDPG309 was cut with BglII/XbaI. Finally, pDPG288 wasrestricted with XbaI/PstI and the potato pinII gene (with intron andterminator) was isolated. After purification, the four fragments wereligated together and transformed into competent DH5α cells. Miniprepanalysis identified the correct clone.

A further plant expression vector that contains the potato pinIIterminator is pDPG343. This plasmid contains the 420-bp 35S promoter,maize Adh1 intron I, a multiple cloning region, and the potato pinIIterminator. pDPG343 was constructed by way of a three-part ligation ofthe following DNAs:

1) 3330 bp EcoRI-PstI fragment of pDPG309, containing the pBluescriptSK(-) and the 420 bp 35S promoter,

2) 580 bp PstI-XbaI fragment of pDPG309, containing the maize Adh1intron 1, and

3) 960 bp XbaI-EcoRI fragment of pDPG331, containing the potato pinIIterminator.

Plasmid pDPG343 contains the following restriction sites between theAdh1 intron I and the potato pinII terminator:

BamHI-SaII-XbaI-SpeI-BamHI

Of the above sites, SpeI in the only unique one in pDPG343. While BamHIcan readily be used for one-step cloning.of trait genes, use of SaII orXbaI will require either three-part (or greater) ligations, or multiplecloning steps.

The DNA fragment encoding the firefly luciferase protein was insertedinto a pUC18-based vector containing the 35S promoter from CauliflowerMosaic Virus, the first intron from the maize Adh1 gene, and thetranscript 7 3' end from Agrobacterium tumefaceiens (provided in theplasmid pCEV1 from Calgene, Inc., Davis, Calif.). This luciferaseexpression vector is referred to as pDPG215, and contains the sameregulatory elements as the bar-expression vector pDPG165.

Two additional luciferase vectors were created, both utilizing intron VIfrom Adh1 (derived from vector pDPG273) fused to firefly luciferase(obtained from vector pDPG215). These elements were inserted into eitherthe pDPG282 (4 OCS inverted-35S) to create pDPG350, or into the pDPG283(4 OCS-35S) backbone to create pDPG351 (bar was excised as a BamHI/NheIfragment and the intron plus luciferase gene inserted). The 4 OCS-35Spromoter has been shown with the uidA gene to give very high levels oftransient expression.

Replication-competent viral vectors are also contemplated for use inmaize transformation. Two wheat dwarf virus (WDV) "shuttle" vectors wereobtained from J. Messing (Rutgers). These vectors, pWI-ll (pDPG386) andpWI-GUS (pDPG387) (FIG. 1 T,U), are capable of autonomous replication incells derived from electroporated maize endosperm protoplasts (Ugaki etal., 1991). Both of these vectors encode a viral replicase and containviral and E. coli origins of replication. In both of these vectors, theviral coat protein coding sequence has been replaced by the neo gene.pDPG387 (pWI-GUS) was created by insertion of a GUS expression cassette(35S-GUS-35S 3') into the BamHI site of pDPG386 (pWI-11).

The expression and integration of marker genes introduced into maizecells on a replicating vector was examined using pDPG387 (pWI-GUS) andBMS cells. Six filters of BMS cells were bombarded with pDPG387/pDPG165(35S-bar-Tr7) and as a control, six filters were bombarded with pDPG128(35S-neo-Tr7)/pDPG165. Tissue from three filters of eachcotransformation was selected on bialaphos (1 mg/l) and tissue from theremaining three filters of each cotransformation was selected onkanamycin (100 mg/l). Selection of kanamycin-resistant colonies fromtissue bombarded with pDPG387 was very inefficient (4 colonies) ascompared to the control treatment in which cells were bombarded withpDPG128/pDPG165 (364 colonies). It also appears that bialaphos selectionwas less efficient for cells bombarded with pDPG387/pDPG165 (59colonies) than it was for the control in which cells were bombarded withpDPG128/pDPG165 (274 colonies).

There are several potential reasons for the low selection efficienciesin bombardments using pDPG387. The neo gene carried by pDPG387 is drivenby the native WDV coat protein promoter; this promoter may not be strongenough to confer kanamycin resistance. Alternatively, the relatively lownumber of bialaphos-resistant colonies recovered from cells bombardedwith pDPG387/pDPG165, infers some sort of negative impact of pDPG387 onthe ultimate selection for expression of a marker gene on a separate,non-replicating vector. The nature of this negative impact, and itsrelationship to the low yield of kanamycin-resistant colonies, iscurrently been investigated.

WDV-bar vectors were constructed to help address the question of theeffect of promoter strength on selection as well as to providereplicating vectors for eventual use with embryogenic cultures. The35S-bar-Tr7 expression cassette was isolated from pDPG165 as anEcoRI/HindIII fragment and protruding ends were filled in with T4 DNApolymerase. This fragment was ligated into pDPG386 (pWI-11) that hadbeen linearized with BamHI, filled in with T4 DNA polymerase, andalkaline phosphatase treated. Vectors containing both orientations ofthe insert were generated and designated pDPG388 and pDPG389 (FIG.1V,W).

A gene encoding the enzyme EPSPS was cloned from Zea mays. Two mutationswere introduced into the amino acid sequence of EPSPS to conferglyphosate resistance, i.e., a substitution of isoleucine for threonineat amino acid position 102 and a substitution of serine for proline atamino acid position 106. Seven plant expression vectors were constructedusing the promoterless mutant maize EPSPS expression vector recievedfrom Rhone Poulenc (pDPG425). The mutant EPSPS gene in this vectorencodes an enzyme with amino acid changes at positions 102 (threonine toisoleucine) and 106 (proline to serine). Seven promoters (±introns) wereused in vector constructions using the mutant maize EPSPS gene. Adescription of the construction of these vectors is presented below.

Four vectors (pDPG434, 436, 441, and 443) were constructed by cloningfour promoters into SmaI-linearized pDPG425. Linearized vectors weretreated with calf alkaline phosphatase to prevent recircularizationprior to ligation. The rice actin promoter and intron were isolated as a1.5 kb HindIII fragment from pDPG421 (pDM302; Cao et al., Plant Cell Rep(1992) 11:586-591). The 35S/adh1 intron I promoter was isolated frompDPG208 as a 0.9 kb HindIII fragment . The 2× Arabidopsis histonepromoter was isolated as a 1.4 kb EcoRI/HindIII fragment from pDPG404.The 2× 35S/Arabidopsis histone promoter was isolated as a 1.8 kbEcoRI/HindIII fragment from pDPG405. The above mentioned promoterfragments were T4 DNA polymerase-treated to create blunt ends prior toligation into SmaI linearized pDPG425 (FIG. 1(Z)). To create pDPG447, a2.1 EcoRI/NcoI α-tubulin/adh1 intron I fragment was isolated frompDPG407 and ligated into EcoRI/NcoI digested pDPG425 (FIG. 1(Z)). Theadh1 promoter and intron I were isolated from pDPG172 (FIG. 1(Y), aderivative of pDPG140 (FIG. 1(X), as a 1.8 kb EcoRI fragment and ligatedinto EcoRI digested, calf alkaline phosphatase-treated pDPG425 (FIG.1(Z)). The resulting plasmid was designated pDPG465. The 2× OCS promoterand adh1 intron VI were isolated from pDPG354 as a 0.6 kb SacI/NcoIfragment and ligated into SacI/NcoI digested pDPG425 (FIG. 1(Z)). Thisplasmids was designated pDPG467. A list of all of the mutant maize EPSPSvectors that were constructed is shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        List of mutant maize EPSPS                                                      plant expression vectors                                                      constructed using pDPG425, and                                                various promoters.                                                              DEKALB Plasmid                                                              Designation Plant Expression Cassette                                       ______________________________________                                        pDPG434       actin - EPSPS - nos                                               pDPG436 35S/adh1 intron I - EPSPS - nos                                       pDPG441 2X Arabidopsis histone - EPSPS - nos                                  pDPG443 2X 35S/Arabidopsis histone - EPSPS -                                   nos                                                                          pDPG447 α-tubulin/adh1 intron I - EPSPS -                                nos                                                                          pDPG465 2X OCS/adh1 intron VI - EPSPS - nos                                   pDPG467 adh1 promoter/adh1 intron I - EPSPS -                                  nos                                                                        ______________________________________                                    

Several vectors were constructed containing genes which may increasestress resistance in transgenic plants, including the mtlD gene from E.coli and the HVA-1 gene from barley. The mannitol operon was originallycloned and characterized by Lee and Saier, 1983. The mtlD gene has beenshown to confer stress resistance on transgenic tobacco plants(Tarczynski, M. C. et al., 1993). A plasmid designated pCD7.5,containing the mtlD gene from this operon (encoding mannitol-1-phosphatedehydrogenase) was obtained from M. Muller, University of Freiburg. Thestructural gene was isolated as a 1500 bp fragment after digestion ofpCD7.5 with NsiI and PstI, and was ligated into a pUC18-based vectorcontaining the 35S promoter from Cauliflower Mosaic Virus, the firstintron from the maize Adh1 gene, and the transcript 7 3' end fromAgrobacterium tumefaceiens. The backbone and regulatory elements wereprepared for this construction by removing the luciferase structuralgene from pDPG215 (35s-AdhI₁ -luc-Tr7 3'; further described in thisdocument), and then religating with an oligonucleotide that created aunique NsiI site between the intron and Tr7 element (this intermediatevector was designated pDPG431). pDPG431 was then linearized using NsiIand the mtlD gene was inserted. The final vector was designated pDPG451(FIG. 1(BB)).

A second expression vector for the mtlD gene was created by removing thebar gene from pDPG182 using SmaI. After blunting the ends of the mtlDgene, it was ligated into the pUC-based vector; between the maizeAdhIpromoter/AdhI₁ intron and the transcript 7 3' end from Agrobacteriumtumefaceiens (provided in pCEV5 from Calgene, Inc., Davis, Calif.). Thisplasmid vector was designated pDPG469.

A gene isolated from barley that encodes a Late Embryogenic Abundantprotein (Dure, L., et al., 1989). (HVa-1) was obtained from Dr. H. D. Ho(Washington University), isolated as an NciI/SphI fragment and clonedinto the polylinker of pCDNA II (Invitrogen, Inc). It was thenreisolated as a BamHI/NsiI fragment and cloned into the polylinker siteof pDPG431 (see above). The result was the pUC-based vector with thefollowing expression unit, 35s-AdhI₁ -HVa1-Tr7 3' (designated pDPG474).

Plasmid constructs designed for increasing the level of lysine in theplant were designed to place a dapA polypeptide-coding sequence,modified to contain a sequence corresponding to one of two maize plastidtransit peptide sequences, under the control of various plant promoterelements. These constructs include the widely-used CaMV 35s promotermaize endosperm-specific promoters from genes encoding either a 27 kD(Z27) or a 10 kD (Z10) zein storage protein, and an embryo-specificpromoter from the maize Globulin1 gene (Glb1), which encodes an abundantembryo storage protein. The transit peptide sequences used herecorrespond to those present in genes encoding either a maize rubiscosmall subunit polypeptide (MZTP) or the native maize DHDPS polypeptide(DSTP). Features of these constructs, along with their lab designations,are as follows:

    ______________________________________                                        pDPG 334               Z10/MZTP/dapA/nos                                         335 Z27/DSTP/dapA/35S                                                         371 35S/MZTP/dapA/nos                                                         372 35S/DSTP/dapA/nos                                                         418 Glb1/DSTP/dapA/Glb1                                                    ______________________________________                                    

Construction of these plasmids was performed as follows:

pDPG371: In this construct, the synthetic pea chloroplast transitpeptide encoding sequence described in U.S. patent application Ser. No.07/204,388 was replaced with a synthetic sequence corresponding to thatencoding a maize chloroplast transit peptide (from a rubisco smallsubunit sequence; GenBank Accession Y00322). Eight oligonucleotides weresynthesized in order to reconstruct the maize ssu transit peptidesequence by the same strategy described for synthesis of the peachloroplast transit peptide sequence (in U.S. patent application Ser.No. 07/204,388). Sequences of these oligonucleotides, in the the finalcontext of the synthetic gene fragment, are as follows (The uppernucleic acid sequence is represented by seq id no:9, the lower nucleicacid sequence by seq id no:10):

    HindIII                    MZTP46                                               MZTP49                                                                        AGCTTGCAGCGAGTACATACATACTAGGCAGCCAGGCAGCCATGGCGCCCACC                             ACGTCGCTCATGTATGTATGATCCGTCGGTCCGTCGGTACCGCGGGTGG                                     MZTP25                      MZTP45                                 -                                                   MZTP51                   GTGATGATGGCCTCGTCGGCCACCGCCGTCGCTCCGTTCCAGGGGCTCAAGTCC                        CACTACTACCGGAGCAGCCGGTGGCGGCAGCGAGGCAAGGTCCCCGAGTTCAGG                                                      MZTP53                                           -                                                 MZTP39                     ACCGCCAGCCTCCCCGTCGCCCGCCGGTCCTCCAGAAGCCTCGGCAACGTCAGC                        TGGCGGTCGGAGGGGCAGCGGGCGGCCAGGAGGTCTTCGGAGCCGTTGCAGTCG                                                                    MZTP54                             -                        SPhI                                                AACGGCGGAAGGATCCGGTGCATG                                                      TTGCCGCCTTCCTAGGCCAC                                                    

The sequence is identical to that of the rubisco ssu gene described inGenBank Y00322, except for the introduction of a HindIII-compatiblesequence generated by the addition of AGCTT to the 5' end of MZTP46 andan A residue to the 3' end of MZTP25. The ATG initiation codon isindicated in bold type, as is the TGC cysteine codon corresponding tothe carboxyterminal residue of the native maize ssu transit peptide.This corresponds to a 47 amino acid transit peptide sequence, from theinitiating methionione to the carboxyterminal cysteine.

Using the same strategy as described for synthesis and reconstruction ofthe pea transit peptide sequence, equimolar amounts of oligonucleotidesMZTP49, 51, 39, 25,45, and 53 were phosphorylated at their 5' ends in apolynucleotide kinase reaction; these were then combined with equimolaramounts of MZTP46 and MZTP54. This mixture was then added to a ligationreaction mixture containing the pGem3 vector (Promega Biotec, Madsion,Wis.) which had previously been cleaved with the restriction enzymesHindIII and SphI to yield plasmid 9106. To fuse the ssu transit peptidesequence (MZTP) to the dapA polypeptide coding sequence, a 1170 bpSphI/EcoRI fragment, corresponding to the dapA/nos 3' cassette presentin plasmid pDAP4201 (in U.S. patent application Ser. No. 07/204,388),was ligated into plasmid 9106 that had been cleaved with SphI and EcoRIto yield pDAP9284. To place this novel construct under control of theplant 35S promoter, a 1300 bp HindIII fragment from pDAP9284, containingthe MZTP/dapA/nos3' cassette, was ligated into the HindIII site ofplasmid 35-227 (U.S. patent application Ser. No. 07/204,388), whichcontains the 35S promoter, to yield plasmid 9305. To facilitate use ofthe 35S/MZTP/dapA/nos3' cassette in future manipulations, the entirecassette was isolated as a 1790 bp ClaI/SmaI fragment from plasmid 9305and inserted into the commercial cloning vector pSP73 (Promega) whichhad been cleaved with ClaI and SmaI. This final construct is designatedpDPG371.

pDPG335: In this construct the synthetic MZTP ssu transit peptidesequence was replaced with DNA encoding the transit peptide sequencefrom the native maize DHDPS enzyme. The plasmid pMDS-1, containing acDNA clone corresponding to maize DHDPS (Frisch et al. Mol. Gen. Genet.228:287-293, 1991), was a gift from B. Gengenbach, University ofMinnesota, Minneapolis. The EcoRI fragment containing the full-lengthmaize DHDPS cDNA was inserted into EcoRI-cleaved pSP73 to yield pMDS73A;this step was performed to facilitate subsequent cloning steps. A 215 bpClaI/XbaI fragment from pMDS73A containing the transit peptide codingregion was inserted into the Clal and XbaI sites of plasmid p35-227,thereby fusing the transit peptide sequence to the 35S promoter sequencecontained in p35-227 and yielding the plasmid pPo135SDTP. The 35S/DSTPcassette was subsequently fused to the dapA coding sequence. This wasperformed by cloning the cassette into the plasmid pHDAP73, which wasconstructed as follows: The 2728 bp hygromycin phosphotransferasecassette from plasmid pHygI1 (U.S. patent application Ser. No.07/204,388) was cloned as an EcoRI/HindIII fragment into the EcoRI andHindIII sites of pSP73, to yield pHyg73. This plasmid was then cleavedwith BglII and ClaI, and the 35S/MZTP/dapA/nos cassette obtained as an1800 bp BglII/ClaI fragment from pDPG371 was inserted to yield pHDAP73.This latter plasmid was then cleaved at the SphI site, at which pointthe MZTP sequence joins the dapA sequence. The SphI 3' overhang wasfilled in by using Klenow fragment to produce a blunt end, and theresultant linear plasmid was subsequently cleaved with BglII, removingthe 35S/MZTP portion from pHDAP73. The 35S/DSTP cassette was isolatedfrom pPo135SDTP by cleavage with XbaI, followed by digestion with mungbean nuclease to remove the resultant 5' overhang. This treatment wasfollowed by BamHI cleavage, which yielded a 674 bp fragment that wasinserted into the cleaved pHDAP73 plasmid in place of the 35S/MZTPsequence. This novel plasmid, containing the cassette 35S/DSTP/dapA/nos,is designated PHDTP. A 1095 bp EcoRI/BamHI fragment, containing theDSTP/dapA region, was cloned into the EcoRI and BamHI sites of thecommercial vector pUC119 (BRL) to yield pDSTP119. Nucleotide sequenceanalysis of this latter clone revealed a 2 bp deletion, apparentlycaused by the cloning process, at the junction of the DSTP and dapAsequences. This mutation was corrected in such a way as to restore theoriginal reading frame and to introduce an additional MaeII restrictionenzyme site as follows:

    ______________________________________                                                       DSTP          dapA                                             ______________________________________                                        target sequence(seq id no:11):                                                                 ATC ACT  |                                                                           TTC ACG GGA                                    CT deleted in pDSTP119(seq id ile thr | phe thr gly                  no:13)                                                                        modify to(seq id no:12): ATC ACG | TTC ACG GCA                     ______________________________________                                    

This manipulation leaves the desired reading frame intact and introducesthe ACGT MaeII site at the junction between the DSTP and dapA sequences.Mutagenesis was accomplished with the reagents supplied in the BioRadMutagene kit by using the oligonucleotide (seq id no:14):

5' CCTTGGCAGCCATCACGTTCACGGGAAGTATTGTC 3'

The resultant plasmid, designated pMae2-1, was cleaved with EcoRI,treated with Klenow polymerase to generate blunt ends, then cleaved withBamHI to yield an 1100 bp fragment consisting of the DSTP/dapA cassette.This fragment was cloned into the Sma and BamHI sites of pZ27Z10 (inU.S. patent application Ser. No. 07/636,089), replacing the Z10 codingregion and placing the DSTP/dapA sequence under control of the Z27promoter and the 35S 3' regulatory region. This final construct isdesignated pDPG335.

pDPG372: To place the DSTP/dapA cassette under control of the 35Spromoter, a 1221 bp ScaI/EcoRI fragment from pHDTP, containing pSP73vector sequences and the 35S promoter, was inserted into the ScaI andEcoRI sites of pMae2-1 to yield p35MDAP. Tto join the nos 3' regulatoryregion to the 35S/DSTP/dapA cassette, an 800 bp fragment of p35MDAP fromthe NdeI site just upstream of the 35S promoter to an NheI site internalto the dapA sequence was inserted into the NdeI and NheI sites ofpDPG371, effectively replacing the 35S/MZTP cassette with the 35S/DSTPsequence. The resultant plasmid, containing the 35S/DSTP/dapA/nos 3'cassette, is designated p35DSD. Subsequent nucleotide sequence analysisof 35DSD revealed the presence of a cloning artifact that introduced anATG translation initiation codon 13 codons upstream of the authenticDSTP initiation codon. This problem was corrected by substituting thisregion of p35DSD with the corresponding region from pDPG335, as follows:a 525 bp fragment from pDPG335, extending from a KpnI site at the 3' endof the 35S promoter to a BstEII site internal to the dapA sequence, wasinserted into KpnI/BstEII-cleaved p35DSD to yield pDPG372, whichcontains a functional 35S/DSTP/dapA/nos cassette.

pDPG334: In this construct, the MZTP/dapA/nos cassette was placed undercontrol of the Z10 promoter region as follows: a 1137 bp HindIII/NcoIfragment from pG10B-H3 (Kirihara et al, Gene 71:359-370, 1988),consisting of the Z10 promoter, was inserted into HindIII/NcoI cleavedpDAP9284, yielding pDPG334 which consists of the Z10 /MZTP/dapA/noscassette.

pDPG418: In this construct, the DSTP/dapA cassette was placed undercontrol of the 5' and 3' regulatory regions of the maize Globulin1(Glb1) gene (Belanger and Kriz Genetics 129:863-872, 1991) as follows: a1050 bp KpnI/PstI fragment from pDPG335, consisting of the DSTP/dapAcassette, was inserted into the KpnI/PstI sites of the Glb1 expressionvector pGEMSV3 (GenBank Accession No. L22295) to yield pDPG418.

Features of introduced genes used for selection of transformed cells aredescribed above. Specific plasmid constructs used in these experimentsare as follows:

    ______________________________________                                        selection                                                                       construct Selectable/screenable marker                                      ______________________________________                                        pDPG 165               bar                                                       231  bar, gus                                                                 283  bar                                                                      355  gus                                                                      363  bar                                                                      366  hpt                                                                      367  hpt                                                                   ______________________________________                                    

Constructs pDPG 165, 231, 283, and 363 are as described above.Constructs pDPG355 and 367 are described in Walters et al., 1992 aspBII221 and pHygI1, respectively. Construct pDPG366 was made bytransferring the hygromycin expression cassette (35S/AdhI1/hpt/nos) frompDPG367 as an EcoRI/HindIII fragment into EcoRI/HindIII cleaved pSP73.

Three plasmids containing the gene encoding the 10 kD zein protein wereconstructed. The plasmid pDPG375 is a 7 kb pUC119 plasmid containing a3.9 kb HindIII fragment of a genomic clone encoding the 10 kD zein(Kirihara et al., 1988). This gene is under control of the nativepromoter, and contains the native 3' sequence. The plasmid pDPG373 is apUC119-based plasmid containing a HindIII-RsaI Z4 (22 kD zein) promoterfragment and an NcoI-EcoRI fragment with the 10 kD zein coding and 3'sequences, and pDPG338 is a pUC119-based plasmid containing the 1.1 kb27 kD zein promoter, 2.3 kb of the 10 kD zein coding and 3' sequences,and a cauliflower mosaic virus (CaMV) 35S poly(A) sequence. Theseplasmids are hereafter designated as the methionine constructs.Selectable marker genes used were hygromycin phosphotransferase (HPT,pHYGI1 also known as pDPG367; 35S promoter::Adh1 intron::HPT codingsequence:: nos poly(A)sequence; Walters et al., 1992) and bar, the geneconferring resistance to the herbicide Basta (35S::bar::Tr7, pDPG165,described earlier in this CIP) or pDPG363 (35S::Adh1::bar::nos). Theplasmid pDPG363 was constructed by inserting a 0.5 kb SmaI fragment withBamHI linkers containing the bar gene into pHYG73, replacing the HPTgene. The plasmid pHYG73 was constructed by insertion of a 2.1 kbEcoRI-HindIII fragment containing the HPT coding sequence from pDPG367(cited above) into pSP73 (Promega). The screenable marker gene ofpBII221, which encodes β-glucuronidase (GUS) was also used(35S::Adh1::GUS::nos). The plasmid pBII221 was constructed by adding a0.75 kb fragment containing the Adh1 intron between the 35S CaMVpromoter and the GUS coding sequence of pBI221 (Clontech; pBI221 ispBI121 in pUC19 rather than pBIN19; Jefferson et al., 1987).

The plasmid pDPG380 contains the entire BalI-EcoRI 711 bp codingsequence of the gene encoding the 19 kD A20 zein and the 0.5 kb 5'sequence encoding the A20 preprotein (reconstructed independently fromthe coding sequence by PCR) in antisense orientation, with 1137 bp ofthe 10 kD zein promoter and 250 bp of nos 3' sequence. The plasmidpDPG340 is a pUC119-based plasmid containing 1137 bp of Z10 promotersequence, a 980 bp XbaI- SacI fragment with the entire Z4 codingsequence in antisense orientation, and 250 bp of nos 3' sequence. Thesegenes are hereafter referred to as antisense constructs. Fortransformation experiments, pDPG165 (35S::bar::Tr7, described elsewherein this CIP), pDPG363 (35S::Adh1::bar::nos, also described elsewhere inthis CIP) or pDPG367 (35S::Adh1::HPT::nos, described elsewhere) wereused as selectable marker genes, and were cobombarded with the antisenseconstructs.

The vector pDPG354 contains an expression cassette for producing Btendotoxin in maize (see FIG. 1(CC) for map). It was constructed tocontain the following DNA:

(i) A promoter, consisting of two ocs enhancers (J. G. Ellis et al.,1987) placed in the reverse orientation and located upstream of the TATAbox derived from the cauliflower mosaic virus (CaMV) 35S promoter (EcoRV site to the transcription start site; H. Guilley et al.,1982; R. Kayet al.1987), located upstream from:

(ii) an intron (intron VI) derived from the maize Adh1 gene (Callis, J.,Fromm, M., Walbot, V. ,1987), a 423 bp AccI-MspI fragment from a genomicclone of the Adh1 gene) that was located upstream from:

(iii) a synthetic Bt gene on a Nco I to Kpn I fragment of DNA coding forthe toxin portion of the endotoxin protein produced by Bacillusthuringiensis subsp.kurstaki strain HD73 (M. J. Adang et al. 1985). Thisgene was synthesized and assembled using standard techniques to containcodons that are more preferred for translation in maize cells. Atranslation stop codon was introduced after the 613th codon to terminatethe translation and allow synthesis of a Bt endotoxin protein consistingof the first 613 amino acids (including the f-met) of the Bt protein(see FIG. 12 for sequence of gene).

(iv) The DNA coding for the Bt protein was followed by a 930 bpRsaI-PstI segment of DNA derived from the potato proteinase II inhibitorgene (G.An et al.,i989).

This expression cassette was inserted into the E.coli plasmidpBluescript SK- (Stratagene, Inc.) and can be made available from theATCC.

The vector pDPG344 was designed to mediate the expression of the tomatoprotease inhibitor II (pin) gene in maize and was constructed to containthe following DNA (see FIG. 1(DD):

(i) 420 bp of DNA derived from the 35S promoter from CaMV locatedupstream of:

(ii) 550 bp derived from the introni of the maize AdhI gene (Callis, J.,Fromm, M. E., Walbot, V., 1987. ) located immediately upstream from

(iii) 470 bp of DNA coding for the cDNA of the tomato protease inhibitorII gene (Graham et al., 1985), followed by:

(iii) 930 bp of DNA derived from the 3' region of the potato proteaseinhibitor II gene (derived from pRJ13; Johnson et al., 1989);

This cassette was assembled on the E.coli plasmid pbluescript SK(-)(Stratagene Inc.) and can be made available from the ATCC.

The plasmid vector pDPG337 (also known as pLK487) consists of an E.colireplicon (pBS+;Stratagene Inc.) containing the following DNA (see FIG.1(EE)):

1. a segment of DNA containing the promoter for the 35S transcriptderived from CaMV, fused at the location of initiation of transcriptionof the 35S transcript to:

2. a segment of DNA (5' ATC TGG CAG CAG AAA AAC AAG TAG TTG AGA ACT AAGAAG AAG AAA 3') (seq id no:15) derived from the untranslated 5' leadersequence to the small subunit of the ribulose biscarboxylase gene ofsoybean (S. L. Berry-Lowe et al.,1982) which is joined at its 3'terminus to:

3. a synthetic Bt gene coding for the endotoxin from Bacillusthuringiensis subsp.kurstaki strain HD73 (see construction of pDPG 354above).

4. The Bt gene was followed by a segment of DNA derived from the 3' of"transcript 7" gene from Agrobacterium tumefasciens (P. Dhaese etal.,1983,).

Samples of E.coli containing pDPG337 can be made available through theATCC.

EXAMPLE 7 Intracellular Targeting of the Bar Gene

As mentioned above, the bar gene codes for an enzyme, PAT, thatinactivates the herbicide phosphinothricin. Phosphinothricin is aninhibitor of both cytoplasmic and chloroplast glutamine synthetases.Current expression vectors target PAT to the cell cytoplasm. Todetermine if there is an advantage to also targeting PAT to thechloroplast, a transit:bar chimeric gene was constructed. A sequenceencoding the rbcS transit peptide and part of the rbcS maturepolypeptide, present on a 300 bp XbaI-BamHI fragment from pDPG226, wascloned adjacent to the bar sequence in pDPG165, to produce pDPG287. Thisresulted in an in-frame fusion of the two protein coding regions, withthe intervening sequence coding for the following amino acids (seq idno:22):

rbcS-Pro-Arg-Gly-Ser-Thr-bar

Protoplasts were electroporated with pDPG287 and assayed for PATactivity. Using proteinase treatment and protection studies, it was thendetermined that the PAT enzyme is sequestered within an organelle,particularly the plastid.

F. Preferred Methods of Delivering DNA to Cells

Preferred DNA delivery systems do not require protoplast isolation oruse of Agrobacterium DNA . There are several potential cellular targetsfor DNA delivery to produce fertile transgenic plants: pollen,microspores, meristems, immature embryos and cultured embryogenic cellsare but a few examples. Germline transformation in maize has not beenpreviously reported.

One of the newly emerging techniques for the introduction of exogenousDNA constructs into plant cells involves the use of microprojectilebombardment. The details of this technique and its use to introduceexogenous DNA into various plant cells are discussed in Klein, 1989,Wang, et al., 1988 and Christou, et al., 1988. One method of determiningthe efficiency of DNA delivery into the cells via microprojectilebombardment employs detection of transient expression of the enzymeβ-glucuronidase (GUS) in bombarded cells. For this method, plant cellsare bombarded with a DNA construct which directs the synthesis of theGUS enzyme.

Apparati are available which perform microprojectile bombardment. Acommercially available source is an apparatus made by Biolistics, Inc.(now DuPont), but other microprojectile or acceleration methods arewithin the scope of this invention. of course, other "gene guns" may beused to introduce DNA into cells.

Several modifications of the microprojectile bombardment method weremade by the inventors. For example, stainless steel mesh screens wereintroduced below the stop plate of the bombardment apparatus, i.e.,between the gun and the cells. Furthermore, modifications to existingtechniques were developed by the inventors for precipitating DNA ontothe microprojectiles.

Another newly emerging technique for the introduction of DNA into plantcells is electroporation of intact cells. The details of this techniqueare described in Krzyzek and Laursen (PCT publication WO 92/12250).Similar to particle bombardment, the efficiency of DNA delivery intocells by electroporation can be determined by using the β-glucuronidasegene. The method of electroporation of intact cells and by extensionintact tissues, e.g., immature embryos, were developed by Krzyzek andLaursen and represent improvements over published procedures. Generationof fertile plants using these techniques were described by Spencer etal. (Spencer, T. M., Laursen, C. M., Krzyzek, R. A., Anderson, P. C.,and Flick, C. E., 1993, Transgenic maize by electroporation ofpectolyase-treated suspension culture cells. Proceedings of the NATOAdvanced Study Institute on Plant Molecular Biology, Molecular-GeneticAnalysis of Plant Metabolism and Development, In Press.) and Laursen etal. (Laursen, C. M., Krzyzek, R. A., Flick, C. E., Anderson, P. C. andSpencer, T. M. Transformation of maize by electroporation of suspensionculture cells. Submitted to Plant Molecular Biology.)

Other methods may also be used for introduction of DNA into plantscells, e.g., agitation of cells with DNA and silicon carbide fibers.

EXAMPLE 8 Microprojectile Bombardment--SC82, SC94 and SC716

For bombardment, friable, embryogenic Type-II callus (Armstrong & Green,1985) was initiated from immature embryos essentially as set forth abovein Examples 1 and 2. The callus was initiated and maintained on N6medium (Chu et al., 1975) containing 2 mg/l glycine, 2.9 g/l L-proline,100 mg/l casein hydrolysate, 13.2 mg/l dicamba or 1 mg/l 2,4-D, 20 g/lsucrose, pH 5.8, solidified with 2 g/l Gelgro (ICN Biochemicals).Suspension cultures initiated from these callus cultures were used forbombardment.

In the case of SC82, suspension culture SC82 was initiated from Type-IIcallus maintained in culture for 3 months. SC82 cells (see Example 2)were grown in liquid medium for approximately 4 months prior tobombardment (see Table 5, experiments #1 and #2). SC82 cells were alsocryopreserved 5 months after suspension culture initiation, storedfrozen for 5 months, thawed and used for bombardment (experiment #6).

In the case of suspension culture SC716 (see Example 1), it wasinitiated from Type-II callus maintained 5 months in culture. SC716cells were cultured in liquid medium for 5 months, cryopreserved for 8months, thawed, and used two months later in bombardment experiments #4and #5. SC94 was initiated from 10 month old Type-II callus; andcultured in liquid medium for 5 months prior to bombardment (experiment#3).

Prior to bombardment, recently subcultured suspension culture cells weresieved through 1000 μm stainless steel mesh. From the fraction of cellclusters passing through the sieve, approximately 0.5 ml packed cellvolume (PCV) was pipetted onto 5 cm filters (Whatman #4) andvacuum-filtered in a Buchner funnel. The filters were transferred topetri dishes containing three 7 cm filters (Whatman #4) moistened with2.5 ml suspension culture medium.

The dish containing the filters with the suspension cells was positioned6 cm below the lexan plate used to stop the nylon macroprojectile. Withrespect to the DNA, when more than a single plasmid was used, plasmidDNA was precipitated in an equimolar ratio onto tungsten particles(average diameter approximately 1.2 μm, GTE Sylvania) using amodification of the protocol described by Klein, et al. (1987). In themodified procedure, tungsten was incubated in ethanol at 65° C. for 12hours prior to being used for precipitation. The precipitation mixtureincluded 1.25 mg tungsten particles, 25 μg plasmid DNA, 1.1 M CaCl₂ and8.7 mM spermidine in a total volume of 575 μl. After adding thecomponents in the above order, the mixture was vortexed at 4° C. for 10min, centrifuged (500×G) for 5 min and 550 μl of supernatant wasdecanted. From the remaining 25 μl of suspension, 1 μl aliquots werepipetted onto the macroprojectile for bombardment.

Each plate of suspension cells was bombarded twice at a vacuum of 28inches Hg. In bombarding the embryogenic suspensions of A188×B73 andA188×B84, 100 μm or 1000 μm stainless steel screens were placed about2.5 cm below the stop plate in order to increase the number of fociwhile decreasing their size and also to ameliorate injury to thebombarded tissue. After bombardment, the suspension cells and thesupporting filter were transferred onto solid medium or the cells werescraped from the filter and resuspended in liquid culture medium.

Cells from embryogenic suspension cultures of maize were bombarded withthe bar-containing plasmid pDPG165 alone or in combination with aplasmid encoding GUS, pDPG208 (FIG. 1). In experiments in which a GUSplasmid was included, two of the filters containing bombarded cells werehistochemically stained 48h post-bombardment. The total number of foci(clusters of cells) per filter transiently expressing GUS was at least1000. In two separate studies designed to quantitate transientlyexpressing cells (using an SC82 (A188×B73) suspension culture), the meannumber of GUS-staining foci per filter was 1472 and 2930. The number ofcells in individual foci that expressed GUS averaged 2-3 (range 1-10).Although histochemical staining can be used to detect cells transformedwith the gene encoding GUS, those cells will no longer grow and divideafter staining. For detecting stable transformants and growing themfurther, e.g., into plants, selective systems compatible with viabilityare required.

EXAMPLE 9 Microprojectile bombardment: AB12

Cell line AB12 was initiated as described in example 4. Themicroprojectile bombardment instrument, microprojectiles and stoppingplates were obtained from Biolistics (Ithaca, N.Y.). Five clumps of 7-to 12-day-old callus, each approximately 50 mg in wet weight, werearranged in a cross pattern in the center of an empty 60 mm×15 mm Petriplate. Plates were stored in a closed container with moist paper towelsthroughout the bombardment process.

Plasmids were coated onto M-10 tungsten particles (Biolistics) asdescribed by Klein et al. (1988, 1989) except that 5 μg of DNA was used,the DNA precipitation onto the particles was performed at 0° C. and thetubes containing the DNA-coated tungsten particles were stored on icethroughout the bombardment process. When both plasmids were used, eachwas present in an amount of 2.5 μg. Control bombardments contained TEbuffer (10 mM Tris, 1 mM EDTA, pH 8.0) with no DNA.

The sample plate tray was placed 5 cm below the bottom of the stoppingplate tray of the microprojectile instrument, with the stopping plateplatform in the slot nearest to the barrel. A 3 mm×3 mm mesh galvanizedsteel screen was placed over the open dish. The instrument was operatedas described by the manufacturer (Biolistics, Inc.), using a gunpowdercharge as the motive force. Each plate of tissue was bombarded once.

EXAMPLE 10 Microprojectile Bombardment--AT824

Suspension culture AT824 (described in example 3) was subcultured tofresh medium 409 2 days prior to particle bombardment. Cells were platedon solid 409 medium 16-24 hours before bombardment (about 0.5 ml packedcell volume per filter). Tissue was treated with 409 medium containing200 mOsm sorbitol (medium 431) for 1 hour prior to bombardment.

DNA was introduced into cells using the DuPont Biolistics PDS1000Heparticle bombardment device.

DNA was precipitated onto gold particles as follows. A stock solution ofgold particles was prepared by adding 60 mg of 1 um gold particles to1000 ul absolute ethanol and incubating for at least 3 hours at roomtemperature followed by storage at -20° C. Twenty to thirty five ulsterile gold particles are centrifuged in a microcentrifuge for 1 min.The supernatant is removed and one ml sterile water is added to thetube, followed by centrifugation at 2000 rpm for 5 minutes.Microprojectile particles are resuspended in 30 ul of DNA solution (30ug total DNA) containing 10 ug each of the following vectors: pDPG165(bar), pDPG344 (tomato proteinase inhibitor II gene), and pDPG354 (B.thuringiensis crystal toxin protein gene). Two hundred twentymicroliters sterile water, 250 ul 2.5 M CaCl₂ and 50 ul spermidine areadded. The mixture is thoroughy mixed and placed on ice, followed byvortexing at 4° C. for 10 minutes and centrifugation at 500 rpm for 5minutes. The supernatant is removed and the pellet resuspended in 600 ulabsolute ethanol. Following centrifugation at 500 rpm for 5 minutes thepellet is resuspended in 36 ul of absolute ethanol .

Ten ul of the particle preparation were dispensed on the surface of theflyer disk and the thanol was allowed to dry completely. Particles wereaccelerated by a helium blast of approximately 1100 psi. One dayfollowing bombardment cells were transferred to liquid medium 409 (10ml). Tissue was subcultured twice per week. During the first week therewas no selection pressure applied.

EXAMPLE 11 Further Optimization of Ballistic Transformation

This example describes the optimization of the ballistic transformationprotocol. Both physical and biological parameters for bombardment havebeen addressed. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells immediately afterbombardment. The prebombardment culturing conditions, such as osmoticenvironment, the bombardment parameters, and the plasmid configurationhave been adjusted to yield the maximum numbers of stable transformants.

Physical Parameters

Gap Distance

The variable nest (macro holder) can be adjusted to vary the distancebetween the rupture disk and the macroprojectile, i.e., the gapdistance. This distance can be varied from 0 to 2 cm. The predictedeffects of a shorter gap are an increase of velocity of both the macro-and microprojectiles, an increased shock wave (which leads to tissuesplattering and increased tissue trauma), and deeper penetration ofmicroprojectiles. Longer gap distances would have the opposite effectsbut may increase viability and therefore the total number of recoveredstable transformants.

The effect of gap distance was investigated by bombarding the E1suspension with pDPG208. Plates were shot in triplicate at gaps of 3, 6,9, and 12 mm. Tissue was assayed for GUS activity and foci were counted.Using a 3 mm gap, GUS foci were the most numerous and well distributedacross the filter. The gas shock wave appeared to be the greatest atthis distance as shown by the degree of tissue splattering. Previousexperiments performed at this gap size have also shown poor tissuerecovery. Gaps of 6 mm and 9 mm showed little to no tissue splattering.GUS foci were well distributed across the filter but were fewer innumber than those in the 3 mm samples. Samples bombarded with a gapdistance of 12 mm showed nearly equivalent numbers of GUS foci as withsample bombarded at 6 mm and 9 mm but they were located almostexclusively at the center of the filter. No tissue splattering wasobserved. Based on these observations, it is suggested that bombardmentsbe conducted with a gap distance of 6 to 9 mm.

Flight Distance

The fixed nest (contained within the variable nest) can be variedbetween 2 and 2 cm in predetermined increments by the placement ofspacer rings to adjust the flight path traversed by the macroprojectile.Short flight paths allow for greater stability of the macroprojectile inflight but reduces the overall velocity of the microprojectiles.Increased stability in flight increases the number of centered GUS foci.Greater flight distances (up to some point) increase velocity but alsoincreases instability in flight.

The effect of the macroprojectile flight path length was investigatedusing E1 suspension cells,. The flight distances tested were 0, 1.0,1.5, and 2.0 cm. Samples were bombarded with pDPG208 GUS vector and wereassayed 48 hours after bombardment for GUS activity. The number of GUSfoci was the greatest at a flight path length of 1.0 cm and least at 0cm. No tissue splattering was observed at 0 cm, very little at 1.0 cm,and greater amount at 1.5 and 2.0 cm. Based on these observations, it isrecommended that bombardments be done with a flight path length of 1.0cm.

Tissue Distance

Placement of tissue within the gun chamber should have significanteffects on microprojectile penetration. Increasing the flight path ofthe microprojectiles will decrease velocity and trauma associated withthe shock wave. A decrease in velocity will also result in shallowerpenetration of the microprojectiles.

Helium Pressure

By manipulation of the type and number of rupture disks, pressure can bevaried between 400 and 2000 psi within the gas acceleration tube.Optimum pressure for stable transformation has been determined to bebetween 1000 and 1200 psi.

Biological Parameters

Culturing conditions and other factors can influence the physiologicalstate of the target cells and may have profound effects ontransformation and integration efficiencies. First, the act ofbombardment could stimulate the production of ethylene which could leadto senescence of the tissue. The addition of antiethylene compoundscould increase transformation efficiencies. Second, it is proposed thatcertain points in the cell cycle may be more appropriate for integrationof introduced DNA. Hence synchronization of cell cultures may enhancethe frequency of production of transformants. Third, the degree oftissue hydration may also contribute to the amount of trauma associatedwith bombardment as well as the ability of the microprojectiles topenetrate cell walls.

It has also been reported that slightly plasmolyzed yeast cells allowincreased transformation efficiencies (Armaleo et al., 1990). It washypothesized that the altered osmotic state of the cells helped toreduce trauma associated with the penetration of the microprojectile.Lastly, the growth and cell cycle stage may be important with respect totransformation.

Osmotic Adjustment

It has been suggested that osmotic pre-treatment could potentiallyreduce bombardment associated injury as a result of the decreased turgorpressure of the plasmolyzed cell. Two studies were done in which E1suspension cells were osmotically adjusted with media supplemented withsorbitol. Cells were plated onto osmotic media 24 hours prior tobombardment. The osmotic values of the media were 200, 400, and 600mOSM/kg. Samples were bombarded with either pDPG208 (GUS) or acoprecipitate of pDPG165 (bar) and pDPG290 (Bt). GUS samples wereassayed and foci were counted and plotted. Cells osmotically adjusted at400 mOSM/kg showed an approximately 25% increase in the number oftransient GUS foci. Samples bombarded with bar/Bt were selected inliquid (2 mg/l bialaphos) and thin plated on medium containing 3 mg/lbialaphos. Cells treated with 600 mOSM/kg medium grew more slowly thancells treated with media of other osmotic strengths in this study.

A second study investigated the effects of short duration osmoticadjustment at 500 mOSM/kg on both transient GUS expression and stabletransformation. The rationale for the short duration of osmoticadjustment was that cells should be plasmolyzed just before bombardment,using longer time periods of pretreatment may allow the cells to adjustto the osmoticum (i.e. re-establishing turgor). The first control wasbombarded (0 min., no new medium) followed by cells pretreated for 45minutes and 90 minutes with 500 mOSM/kg medium with either pDPG208 orpDPG165 with pDPG290. Since the pretreatment required media changes(i.e. fresh 500 mOSM/kg media), a set of controls were also washed usingfresh medium without the osmoticum. After bombardment the cells were puton to solid medium to recover overnight followed by resuspension inliquid medium. After one week, liquid selection was started using 2μg/ml bialaphos. Cells were plated on 3 μg/ml bialaphos at 0.1 ml PCVeleven days after bombardment. Transient GUS activity was assayed 48hours after bombardment.

The number of cells transiently expressing GUS increased followingsubculture into both fresh medium and osmotically adjusted medium.Pretreatment times of 90 minutes showed higher numbers of GUS expressingfoci than shorter times. Cells incubated in 500 mOSM/kg medium for 90minutes showed an approximately 3.5 fold increase in transient GUS focithan the control.

Plasmid Configuration

In some instances it will be desirable to deliver DNA to maize cellsthat does not contain DNA sequences necessary for maintenance of theplasmid vector in the bacterial host, e.g., E. coli, such as antibioticresistance genes, including but not limited to ampicillin, kanamycin,and tetracycline resistance, and prokaryotic origins of DNA replication.In one such experiment the 4.4 kb HindIII fragment of pDPG325 containingthe bar expression cassette and 2 kb of the uidA expression cassette(structural gene and 3' end) were purified by gel electrophoresis on a1.2% low melting temperature agarose gel. The 4.4 kb DNA fragment wasrecovered from the agarose gel by melting gel slices in a 6-10 foldexcess of Tris-EDTA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 70-72°C.); frozen and thawed (37° C.); and the agarose pelleted bycentrifugation. A Qiagen Q-100 column was used for purification of DNA.For efficient recovery of DNA it was necessary to reduce the flow rateof the column to 40 ml/hr. Isolated DNA fragments can be recovered fromagarose gels using a variety of electroelution techniques, enzymedigestion of the agarose, or binding of DNA to glass beads (e.g., GeneClean). In addition HPLC and/or use of magnetic particles may be used toisolate DNA fragments. This DNA was delivered to AT824 cells usingmicroprojectile bombardment. Twenty four transformants were recoveredfollowing selection on bialaphos containing culture medium. Notransformants contained the ampicillin resistance gene or origin of DNAreplication present in the plasmid vector. R₀ plants have been producedfrom 11 of these transformed cell lines. Fertility has been demonstratedin plants from ten transformants and R₁ seed has been planted in fieldtests.

As an alternative to isolation of DNA fragments a plasmid vector can bedigested with a restriction enzyme and this DNA delivered to maize cellswithout prior purification of the expression cassette fragment. In oneexperiment pDPG165 was digested with EcoRI and HindIII. This digestionproduces an approximately 1900 base pair fragment containing the35S-bar-Tr7 expression cassette and an approximately 2600 base pair DNAfragment containing the ampicillin resistance gene and bacterial originof DNA replication. This DNA was delivered to AT824 cells usingmicroprojectile bombardment and 2/9 transformants (22%) isolated did notcontain the ampicillin resistance gene. In a second experiment pDPG165digested with restriction enzymes as described above was delivered toAT824 cells via electroporation. Eight of twenty four transformants(33%) recovered lacked the ampicillin resistance gene. Plantregeneration is in progress from transformants lacking the ampicillinresistance gene that were produced in these two experiments.

EXAMPLE 12 Bombardment of Immature Embryos

Immature embryos (1.2-2.0 mm in length) were excised fromsurface-sterilized, greenhouse-grown ears of Hi-II 11-12 dayspost-pollination. The Hi-II genotype was developed from an A188×B73cross for high frequency development of type II callus from immatureembryos (Armstrong et al., 1991). Approximately 30 embryos per petridish were plated axis side down on a modified N6 medium containing 1mg/l 2,4-D, 100 mg/l casein hydrolysate, 6 mM L-proline, 0.5 g/l2-(N-morpholino)ethanesulfonic acid (MES), 0.75 g/l MgCl₂, and 2%sucrose solidified with 2 g/l Gelgro, pH 5.8 (#735 medium) Embryos werecultured in the dark for two days at 24° C.

Approximately four hours prior to bombardment, embryos were transferredto the above culture medium with the sucrose concentration increasedfrom 3% to 12%. When embryos were transferred to the high osmoticummedium they were arranged in concentric circles on the plate, starting 2cm from the center of the dish, positioned such that their coleorhizalend was orientated toward the center of the dish. Usually two concentriccircles were formed with 25-35 embryos per plate.

Preparation of gold particles carrying plasmid DNA was described inexample 10. Particles were prepared containing 10 ug pDPG215(luciferase), pDPG415 (Bt), and pDPG417 (bar) or 30 ug pDPG265containing the maize R and C1B genes for anthocyanin biosynthesis.

The plates containing embryos were placed on the third shelf from thebottom, 5 cm below the stopping screen. The 1100 psi rupture discs wereused. Each plate of embryos was bombarded once. A total of 420 embryoswere bombarded on 14 plates with the luciferase, bar, and Bt genes.Embryos were allowed to recover overnight on high osmotic strengthmedium prior to initiation of selection. A set of plates was alsobombarded with the C1B vector pDPG265. Red spots representing transientexpression of anthocyanin pigments are observed 24 hours after DNAintroduction.

EXAMPLE 13 Electroporation Experiment EP413: Stable Transformation ofSC716 and AT824 Cells Using pDPG165 and pDPG208

Maize suspension culture cells were enzyme treated and electroporatedusing conditions described in Krzyzek and Laursen (PCT Publication WO92/12250). SC716 or AT824 suspension culture cells, three days postsubculture, were sieved through 1000 μm stainless steel mesh and washed,1.5 ml packed cells per 10 ml, in incubation buffer (0.2 M mannitol,0.1% bovine serum albumin, 80 mM calcium chloride, and 20 mM2-(N-morpholino)-ethane sulfonic acid, pH 5.6). Cells were then treatedfor 90 minutes in incubation buffer containing 0.5% pectolyase Y-23(Seishin Pharmaceutical, Tokyo, Japan) at a density of 1.5 ml packedcells per 5 ml of enzyme solution. During the enzyme treatment, cellswere incubated in the dark at approximately 25° C. on a rotary shaker at60 rpm. Following pectolyase treatment, cells were washed once with 10ml of incubation buffer followed by three washes with electroporationbuffer (10 mM HEPES, 0.4 mM mannitol). Cells were resuspended inelectroporation buffer at a density of 1.5 ml packed cells in a totalvolume of 3 ml.

Linearized plasmid DNA, 100 ug of EcoRI digested pDPG165 and 100 ug ofEcoRI digested pDPG208, was added to 1 ml aliquots of electroporationbuffer. The DNA/electroporation buffer was incubated at room temperaturefor approximately 10 minutes. To these aliquots, 1 ml of suspensionculture cells/electroporation buffer (containing. approximately 0.5 mlpacked cells) were added. Cells and DNA in electroporation buffer wereincubated at room temperature for approximately 10 minutes. One half mlaliquots of this mixture were transferred to the electroporation chamber(Puite, 1985) which was placed in a sterile 60×15 mm petri dish. Cellswere electroporated with a 70, 100, or 140 volt (V) pulse dischargedfrom a 140 microfarad (μf) capacitor.

Approximately 10 minutes post-electroporation, cells were diluted with2.5 ml 409 medium containing 0.3 M mannitol. Cells were then separatedfrom most of the liquid medium by drawing the suspension up in a pipet,and expelling the medium with the tip of the pipet placed against thepetri dish to retain the cells. The cells, and a small amount of medium(approximately 0.2 ml) were dispensed onto a filter (Whatman #1, 4.25cm) overlaying solid 227 medium (Table 1) containing 0.3 M mannitol.After five days, the tissue and the supporting filters were transferredto 227 medium containing 0.2 M mannitol. After seven days, tissue andsupporting filters were transferred to 227 medium without mannitol.

EXAMPLE 14 Electroporation of Immature Embryos

Immature embryos (0.4-1.8 mm in length) were excised from asurface-sterilized, greenhouse-grown ear of the genotype H99 11 dayspost-pollination. Embryos were plated axis side down on a modified N6medium containing 3.3 mg/l dicamba, 100 mg/l casein hydrolysate, 12 mML-proline, and 3% sucrose solidified with 2 g/l Gelgro®, pH 5.8 (#726medium), with about 30 embryos per dish. Embryos were cultured in thedark for two days at 24° C.

Immediately prior to electroporation, embryos were enzymatically treatedwith 0.5% Pectolyase Y-23 (Seishin Pharmaceutical Co.) in a buffercontaining 0.2 M mannitol, 0.2% bovine serum albumin, 80 mM calciumchloride and 20 mM 2-(N-morpholino)-ethane sulfonic acid (MES) at pH5.6. Enzymatic digestion was carried out for 5 minutes at roomtemperature. Approximately 140 embryos were treated in batch in 2 ml ofenzyme and buffer. The embryos were washed two times with 1 ml of 0.2 Mmannitol, 0.2% bovine serum albumin, 80 mM calcium chloride and 20 mMMES at pH 5.6 followed by three rinses with electroporation bufferconsisting of 10 mM 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES) and 0.4 M mannitol at pH 7.5. For the electroporations, thefinal rinse of electroporation buffer was removed and the embryos wereincubated with 0.33 mg/ml linearized pDPG165, 0.33 mg/ml supercoiledpDPG215 and 0.33 mg/ml linearized pDPG344 in electroporation buffer. Onehalf ml aliquots of DNA in electroporation buffer and twenty embryoswere transferred to the electroporation chamber that was placed in asterile 60×15 mm Petri dish. An electrical pulse was passed through thecells from a 500 μf capacitor that was charged to 100 volts (400 V/cmfield strength, 160 ms pulse decay time; exponential pulse).

Immediately following electroporation, embryos were diluted 1:10 with726 medium containing 0.3 M mannitol. Embryos were then transferred toGelgro® solidified 726 medium containing 0.3 M mannitol. Embryos wereincubated in the dark at 24° C. After five days embryos were transferredto Gelgro solidified 726 medium containing 0.2 M mannitol. Two dayslater embryos were transferred to selection medium.

EXAMPLE 15 DNA Delivery Using Silicon Carbide Fibers

Kaeppler et al. 1990 reported transformation of tobacco and BMSsuspensions using fibers having a size of 0.6×10 to 80 μm.Transformations were accomplished by vortexing the silar fibers togetherwith cells in a DNA solution. DNA passively enters as the cell arepunctured. It is contemplated that fibers and/or particles of othertypes would also be useful. The suspension culture SC82 was tested fortransformability using the silicon carbide (silar) transformation methoddescribed by Kaeppler et al. The initiation of cell line SC82 isdescribed in example 2.

A 2% mixture of silar in absolute ethanol was prepared. Microfuge tubeswere prepared (one per sample) by pipetting 80 μl of silar into eachtube. The fibers were pelleted and the ethanol removed. Samples werethen washed with sterile water, pelleted, and the water removed. PlasmidDNA (25 μl of 1 mg/ml) was added to each tube. Tissue samples wereprepared by adding 0.25 ml PCV of cells to a second set of microfugetubes. Cells were pelleted and the medium removed. A 100 μl aliquot offresh medium was next added to each tissue sample. The silar/DNA mixturewas resuspended and added to the cells. The transformation was carriedout by inverting the microfuge tubes and vortexing for 10 secondsfollowed by placing the tube upright and vortexing for an additional oneminute. Samples were then removed and cultured in small petri disheswith 3 ml of medium. Transient GUS activity was observed two days afterDNA delivery.

In a second experiment with SC82 a bead beater or a vortex was utilizedto agitate samples. Samples were prepared as described above. Twosamples were vortexed and the remaining samples were agitated with thebead beater for 5, 15, and 30 seconds at either the low or the highsetting. The bead beater method showed a substantial increase intransient GUS activity as compared to vortexed samples. In all casessamples agitated on the "slow" setting of the bead beater showed highertransient GUS activity and less tissue damage than those on the "high"setting.

Three samples were vortexed in the presence of pDPG165 for selection ofstably transformed cell lines. Each sample (0.25 ml PCV) was returned toa 25 ml flask and grown to 2 ml PCV at which time it was used asinoculum for a 125 ml flask. These were passaged once before platingonto 1 mg/l bialaphos containing medium. Cultures were plated ontofilters at differing densities. Flask #1 was plated at 0.5 ml PCV, flask#2 at 0.25 ml PCV and flask #3 at 0.1 ml PCV. Filters containing cellswere transferred three times onto bialaphos containing medium. Tissuearising from flask #1 was quite dense at the end of this phase ofselection, tissue was scraped from a filter, divided into fourths andspread onto solidified selective medium. No transformants were recoveredfrom this method. Tissue arising from flasks #2 and #3 were selected asdescribed in example 8 and 18 bialaphos resistant transformants wererecovered. No plant regeneration was attempted.

G. Identification of Transformed Cells Using Selectable Markers

It is believed that DNA is introduced into only a small percentage ofcells in any one experiment. In order to provide a more efficient systemfor identification of those cells receiving DNA and integrating it intotheir genomes, therefore, one may desire to employ a means for selectingthose cells that are stably transformed. One exemplary embodiment ofsuch a method is to introduce into the host cell, a marker gene whichconfers resistance to some normally inhibitory agent, e.g. an antibioticor herbicide. The potentially transformed cells are then exposed to theagent. In the population of surviving cells are those cells whereingenerally the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA. Usingembryogenic suspension cultures, stable transformants are recovered at afrequency of approximately 1 per 1000 transiently expressing foci. Aspecific embodiment of this procedure is shown in Example 17.

One of the difficulties in cereal transformation, e.g., corn, has beenthe lack of an effective selective agent for transformed cells, fromtotipotent cultures (Potrykus, 1989). Stable transformants wererecovered from bombarded nonembryogenic Black Mexican Sweet (BMS) maizesuspension culture cells, using the neo gene and selection with theaminoglycoside, kanamycin (Klein, 1989). This approach, while applicableto the present invention, is not preferred because many monocots areinsensitive to high concentrations of aminoglycosides (Dekeyser et al.,1989; Hauptmann et al., 1988). The stage of cell growth, duration ofexposure and concentration of the antibiotic, may be critical to thesuccessful use of aminoglycosides as selective agents to identifytransformants (Lyznik et al., 1989; Yang et al., 1988; Zhang et al.,1988). For example, D'Halluin et al. (1992) demonstrated that using theneo gene and selecting with kanamycin transformants could be isolatedfollowing electroporation of immature embryos of the genotype H99 ortype I callus of the genotype PA91. In addition, use of theaminoglycosides, kanamycin or G418, to select stable transformants fromembryogenic maize cultures, in the inventors' experience, often resultsin the isolation of resistant calli that do not contain the neo gene.

One herbicide which has been suggested as a desirable selection agent isthe broad spectrum herbicide bialaphos. Bialaphos is a tripeptideantibiotic produced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism (Ogawa et al., 1973). SyntheticPPT, the active ingredient in the herbicides Basta® or Ignite® is alsoeffective as a selection agent. Inhibition of GS in plants by PPT causesthe rapid accumulation of ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide resistance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorgaism this enzyme acetylates the free amino group of PPT preventingauto-toxicity (Thompson et al., 1987). The bar gene has been cloned(Murakami et al., 1986; Thompson et al., 1987) and expressed intransgenic tobacco, tomato and potato plants (De Block, 1987) andBrassica (De Block, 1989). In previous reports, some transgenic plantswhich expressed the resistance gene were completely resistant tocommercial formulations of PPT and bialaphos in greenhouses.

PCT Application No. WO 87/00141 refers to the use of a process forprotecting plant cells and plants against the action of glutaminesynthetase inhibitors. This application also refers to the use of suchof a process to develop herbicide resistance in determined plants. Thegene encoding resistance to the herbicide BASTA (Hoechst,phosphinothricin) or Herbiace (Meiji Seika, bialaphos) was said to beintroduced by Agrobacterium infection into tobacco (Nicotiana tabacum cvPetit Havan SR1), potato (Solanum tuberosum cv Benolima) and tomato(Lycopersicum esculentum) and conferred on plants resistance toapplication of herbicides.

Another herbicide which is useful for selection of transformed celllines in the practice of this invention is the broad spectrum herbicideglyphosate. Glyphosate inhibits the action of the enzyme EPSPS which isactive in the aromatic amino acid biosynthetic pathway. Inhibition ofthis enzyme leads to starvation for the amino acids phenylalanine,tyrosine, and tryptophan and secondary metabolites derived thereof. U.S.Pat. No. 4,535,060 describes the isolation of EPSPS mutations whichinfer glyphosate resistance on the Salmonella typhimurium gene forEPSPS, aroA. The EPSPS gene was cloned from Zea mays and mutationssimilar to those found in a glyphosate resistant aroA gene wereintroduced in vitro. The mutant gene encodes a protein with amino acidchanges at residues 102 and 106. Although these mutations conferresistance to glyphosate on the enzyme EPSPS it is anticipated thatother mutations will also be useful.

An exemplary embodiment of vectors capable of delivering DNA to planthost cells is the plasmid, pDPG165 and the vectors pDPG433, pDPG434,pDPG435, and pDPG436. The plasmid pDPG165 is illustrated in FIG. 1A and1C. A very important component of this plasmid for purposes of genetictransformation is the bar gene which encodes a marker for selection oftransformed cells exposed to bialaphos or PPT. Plasmids pDPG434 andpDPG436 contain a maize EPSPS gene with mutations at amino acid residues102 and 106 driven by the actin promoter and 35S promoter-Adh1 intron Irespectively. A very important component of these plasmids for purposesof genetic transformation is the mutated EPSPS gene which encodes amarker for selection of transformed cells.

EXAMPLE 16 Selection of Bar Transformants Using Bialaphos in the CellLine SC82 Following Particle Bombardment

The suspension culture (designated SC82) used in the initial experiments(see Example 8) was derived from embryogenic Type-II callus of A188×B73.Following bombardment (see Example 8), cells on filters were resuspendedin nonselective liquid medium, cultured for 1 to 2 weeks and transferredto filters overlaying solid medium containing 1 or 3 mg/l bialaphos. Thedegree of inhibition of tissue growth during selection was dependentupon the density of the cells on the filter and on the concentration ofbialaphos used. At the density plated (0.5 PCV/filter), growth of cellscultured on 1 mg/l bialaphos was only partially inhibited (˜30-50% ofnonselected growth) and after 3 to 4 weeks much of this tissue wastransferred as discrete clumps (˜5 mm in diameter) to identical medium.On medium containing 3 mg/l bialaphos, the growth of cells on theoriginal selection filter was severely inhibited (˜10% of nonselectedgrowth) and selection was carried out without removing the tissue fromthe original filter.

Using either selection protocol (1 or 3 mg/l bialaphos), resistant cellcolonies emerged on the selection plates of SC82 bombarded with pDPG165approximately 6 to 7 weeks after bombardment (FIG. 2A).Bialaphos-resistant calli were maintained and expanded on selectionmedium. Much of this tissue was embryogenic (FIG. 2B). No colony growthoccurred on plates to which cells were added from untransformedsuspension cultures. These were controls which confirm the predictionthat cells without the bar gene are not resistant to bialaphos.

Colonies on solid supports are visible groups of cells formed by growthand division of cells plated on such support. Colonies can be seen inFIG. 2A on a petri dish. In this figure, the cells capable of growth arethose that are resistant to the presence of the herbicide bialaphos,said resistance resulting from integration and expression of the bargene. Exposure of cells was to 1 mg/l bialaphos. FIG. 2B is amagnification showing the morphology of one bialaphos-resistant culturemaintained on selection media indicating that growth is embryogenic.

As a confirmation that the cells forming the colonies shown in FIG. 2had indeed incorporated the bar gene and were expressing it,bialaphos-resistant callus lines were analyzed for activity of the bargene product, phosphinothricin acetyl transferase (PAT), by thin-layerchromatography. Protein extracts from eleven callus lines (E1-11)isolated from SC82 bombardment experiments contained PAT activity asshown in FIG. 3 and activity levels varied approximately 10-fold amongthe isolates.

Still further and more direct confirmation of the presence of the bargene was obtained by analysis of the genomic DNA of potentialtransformants by DNA gel blots (FIG. 4). The sources of DNA which wereelectrophoresed through the gel were the bialaphos-resistant calluslines designated E1-E11 and a non-selected control, E0. (FIG. 1indicates the cleavage sites of those enzymes within the bar geneplasmid). After the DNA was electrophoresed through the gel andtransferred to nylon membranes, the resulting blot was hybridized with a³² P-labeled bar gene sequence from the plasmid pDPG165. Theradioactivity used per blot as approximately 25×10⁶ Cerenkov cpm. Thelane in FIG. 4 designated "1" and "5" copies contain 1.9 and 9.5 pgrespectively of the 1.9 kb bar expression unit released from the plasmidpDPG165 by application of the EcoRI and HindIII enzymes; these amountsrepresent about 1 and 5 copies per diploid genome.

Genomic DNA from all eleven bialaphos-resistant isolates containedbar-hybridizing sequences as shown in FIG. 4. The hybridization in allisolates to a fragment migrating slightly larger than 2 kb may be due tocontaminating pUC19 sequences contained in this bar probe preparation;no such hybridization occurred in subsequent experiments using the samegenomic DNA and a different preparation of the bar probe. Hybridizationto a 1.9 kb fragment in eight of the eleven isolates indicated thatthese isolates contained intact copies of the 1.9 kb bar expressionunit. The estimated copy numbers of the intact unit ranged from one ortwo (E1, E7, E8, E10, E11) to approximately 20 (E3, E4, E6).Hybridization with the bar probe in isolates E2 and E5 occurred only toa single, higher molecular weight fragment (˜3 kb).

To establish that the PAT coding sequence was intact in isolates E2 andE5, genomic DNA was digested with SmaI, which releases a 559 bp fragmentcontaining the PAT structural gene (FIG. 1A), and subjected to DNA gelblot analysis using ³² P-labeled bar. This analysis confirmed thepresence of a single intact copy of bar. Expression of PAT in theseisolates may not be dependent on the 35S promoter or the Tr7 3' end. Thehybridization patterns of some of the isolates were identical (E2 andE5; E7 and E8; E3, E4, and E6); therefore, it is probable that someisolates did not arise from independent transformation events butrepresent transformants that were separated during selection.

Seven hybridization patterns were unique, likely representing sevenindependent single-cell transformation events. The patterns andintensities of hybridization for the seven transformants were unchangedduring four months in culture, providing evidence for the stability ofthe integrated sequences. The seven independent transformants werederived from two separate bombardment experiments. Four independenttransformants representing isolates E2/E5, E3/E4/E6, E1 and E7/E8, wererecovered from a total of four original filters from bombardmentexperiment #1 and the three additional independent transformants, E9,E10, and E11, were selected from tissue originating from six bombardedfilters in experiment #2. These data are summarized in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    Summary of Maize Transformation Experiments                                                  # of   # with                                                       Independent Intact # with                                                    # of bar bar GUS # with Cointegration Coexpression                          Exp. Culture Filters Transformants Expression Coding GUS Frequency                                                      Frequency                           # Bombarded Bombarded Recovered Units Sequence Activity (%) (%)             __________________________________________________________________________    1   SC82 4     4      3     n.a                                                 2 SC82 6 3 2 n.a.                                                             3 SC94 10  8 6 n.a.                                                           4 SC716* 8 13  8 11  3 85 23                                                  5 SC716* 8 7 4 6 1 86 14                                                      6 SC82* 4 19  17  13  3 68 16                                                  TOTALS 40  54  40  30  7 77 (30/39) 18 (7/39)                              __________________________________________________________________________     *culture reinitiated from cryopreserved cells                                 n.a. not applicable; only pDPG165 DNA used or cotransformation analysis       not done                                                                 

Studies with other embryogenic suspension cultures produced similarresults. Using either an SC82 culture that was reinitiated fromcryopreserved cells (experiment #6) or an A188×B84 (SC94) suspensionculture (experiment #3), numerous independent transformants wererecovered (19 and 18 respectively; Table 5). All transformants containedthe bar gene and expressed PAT. The copy number of bar-hybridizingsequences and levels of PAT expression were comparable to the studiesdescribed above.

EXAMPLE 17 Integration of the Bar Gene into Cell Lines Derived from theSC716 Suspension Culture

Bombardment studies and subsequent analyses were also performed on theA188×B73 suspension culture, termed SC716 (see Example 1). The resultanttransformed plant cells were analyzed for integration of bar genes. Tocarry out this analysis, genomic DNA was obtained from R1-R21 isolates;6 μg of DNA was digested with the restriction endonucleases EcoRI andHindIII, and DNA gel blot analysis was performed using the bar gene asprobe. In FIG. 5, molecular weights in kb are shown to the right andleft. The untransformed control is designated "R0," and the last columnto the right contains the equivalent of two copies of the bar geneexpression unit per diploid genome. For the DNA load used, two copiesthe bar expression unit per diploid genome is 5.7 pg of the 1.9 kbEcoRI/Hind fragment from the plasmid pDPG165. The DNA separated on thegel blot was hybridized to a ³² P-labeled bar probe. The label activityin the hybridization was approximately 10×10⁶ Cerenkov cpm. In A, thepresence of an intact bar expression unit is inferred from thehybridization of the bar probe to a 1.9 kb band in the gel.

EXAMPLE 18 Assays for Integration and Expression of GUS

SC716 transformants discussed in Example 17, were further analyzed forintegration and expression of the gene encoding GUS. As determined byhistochemical assay, four of the SC716 transformants (R5, R7, R16, andR21) had detectable GUS activity 3 months post-bombardment. Expressionpatterns observed in the four coexpressing callus lines varied. Thenumber of cells with GUS activity within any given transformant sampledranged from ˜5% to ˜90% and, in addition, the level of GUS activitywithin those cells varied. The cointegration frequency was determined bywashing the genomic blot hybridized with bar (FIG. 5A) and probing with³² P-labeled GUS sequence as shown in FIG. 5B. EcoRI and HindIII, whichexcise the bar expression unit from pDPG165, also release from pDPG208 a2.1 kb fragment containing the GUS coding sequence and the nos 3' end(FIG. 1B).

Seventeen of the independent bar transformants contained sequences thathybridized to the GUS probe; three, R2, R14 and R19 did not.Transformants in which GUS activity was detected (R5, R7, R16 and R21)had intact copies of the 2.1 kb EcoRI/HindIII fragment containing theGUS structural gene (FIG. 5B). Transformants that contained largenumbers of fragments that hybridized to bar (R1, R5, R21) also containedcomparable number of fragments that hybridized to the gene encoding GUS(FIGS. 5A and B). This observation is consistent with those reportedusing independent plasmids in PEG-mediated transformation of A188×BMSprotoplasts (Lyznik et al., 1989) and in studies conducted by theinventors involving bombardment-mediated transformation of BMSsuspension cells.

EXAMPLE 19 Transformation of Cell Line AT824 Using Bialaphos SelectionFollowing Particle Bombardment--Selection in Liquid Medium

The suspension culture (designated AT824) used in this experiment wasderived from an elite B73-derived inbred (described in example 3). Theculture was maintained in medium 409. Four filters were bombarded asdescribed in example 10.

Following one week culture in liquid medium 409 without selectionpressure, tissue was transferred to fliquid medium 409 containing 1 mg/Lbialaphos. Cells were transferred twice per week into fresh mediumcontaining 1 mg/L bialaphos for two weeks. Tissue was thin planted 3weeks following bombardment at a concentration of 0.1 ml packed cellvolume per petri dish containing medium 425 (with 3 mg/L bialaphos).Transformants were identified as discreet colonies 6 weeks followingbombardment. It is the experience of the inventors that all cell linesthat grow on 3 mg/L bialaphos contain the bar gene. Fifty transformedcell lines were recovered from this experiment. Twenty four of thesecell lines contained the Bt gene.

EXAMPLE 20 Transformation of Cell Line AT824 Using Bialaphos SelectionFollowing Particle Bombardment--Solid Medium Selection

Cells in experiment S10 were bombarded as described in example 10 exceptthe gold particle-DNA preparation was made using 25 ul pDPG319 DNA (bargene and aroA expression cassette containing the α-tubulin promoter).Following particle bombardment cells remained on solid 279 medium in theabsence of selection for one week. At this time cells were removed fromsolid medium, resuspended in liquid 279 medium, replated on Whatmanfilters at 0.5 ml PCV per filter, and transferred to 279 mediumcontaining 1 mg/L bialaphos. Following one week, filters weretransferred to 279 medium containing 3 mg/L bialaphos. One week later,cells were resuspended in liquid 279 medium and plated at 0.1 ml PCV on279 medium containing 3 mg/L bialaphos. Nine transformants wereidentified 7 weeks following bombardment.

EXAMPLE 21 Transformation of Cell Line ABT4 Using Bialaphos FollowingParticle Bombardment

Initiation of cell line ABT4 is described in example 4. ABT4 wasmaintained as a callus culture. At the time of subculture, tissue wasscraped off the solid culture medium and resuspended in 20 mls of 708medium containing 0.2M mannitol. Tissue was dispersed with a large bore10 ml pipette by picking up and dispensing several times until one couldpickup 0.5 ml packed cell volume (PCV) for subculture to fresh solid 708medium. Prior to bombardment three week old 708 maintenance cultures ofABT4 were transferred from solid medium to 20 mls 708+0.2M mannitol and0.5 ml PCV was plated on glass fiber filters over 708+0.2M mannitolmedium. Cultures were allowed to plasmolyze for 2-4 hours prior tobombardment. At the time of bombardment tissue on a glass fiber filterwas placed on top of 3 filter papers moistened with 2.5 mls of 708+0.2Mmannitol. Six ul of DNA/gold particles (described in example 10) wasplaced on flyers prior to bombardment with the Dupont BiolisticsPDS1000He particle delivery device. Particles were accelerated by a 1100psi blast of helium gas. Following bombardment tissue was returned to708+0.2M mannitol and allowed to recover for 2-5 days. Selection beganat this point by moving the tissue/filter to 708+1 mg/L bialaphos for 12days. At this time tissue was transferred to 30-40 ml 708+0.5 mg/Lbialaphos, dispersed, and thin plated at 0.05 to 0.10 PCV on 708+0.5mg/L bialaphos solid medium. Transformants were identified 5-12 weeksfollowing thin plating. Following identification transformants weremaintained on 708+3 mg/L bialaphos.

EXAMPLE 22 Transformation of Immature Embryos of the Genotype Hi-IIUsing Bialaphos as a Selective Agent Following Particle Bombardment

Immature embryos of the genotype Hi-II were bombarded as described inexample 12. Embryos were allowed to recover on high osmoticum medium(735, 12% sucrose) overnight (16-24 hours) and were then transferred toselection medium containing 1 mg/l bialaphos (#739, 735 plus 1 mg/lbialaphos or #750, 735 plus 0.2M mannitol and 1 mg/l bialaphos). Embryoswere maintained in the dark at 24° C. After three to four week on theinitial selection plates about 90% of the embryos had formed Type IIcallus and were transferred to selective medium containing 3 mg/lbialaphos (#758). Responding tissue was subcultured about every twoweeks onto fresh selection medium (#758). Nineteen transformants wereidentified six to eight weeks after bombardment. Fifteen of nineteentransformants contained the B. thuringiensis (Bt) crystal toxin gene.Plants have been regenerated from one transformant containing the Btgene and transferred to soil in the greenhouse. Regeneration of plantsfrom remaining lines containing the Bt gene is in progress.

EXAMPLE 23 Transformation of AT824 and SC716 Using Bialaphos SelectionFollowing Electroporation

Cells of AT824 and SC716 were electroporated and allowed to recover fromelectroporation as described in example 13. Five days later, tissuegrowing on filters was removed from the filter and transferred as clumps(approximately 0.5 cm in diameter) to the surface of solid selectionmedium. The selection medium consisted of 227 medium supplemented with 1mg/L bialaphos. Three weeks later, slowly growing tissue wastransferred, as 0.5 cm clumps, to 227 medium containing 3 mg/Lbialaphos. Three to four weeks later, callus sectors that continued togrow were transferred to fresh 227 medium containing 3 mg/L bialaphos.Callus lines that continued to grow after this subculture wereconsidered to be transgenic and perpetuated further, by transfer tofresh selection medium every two weeks. One SC716 and seven AT824 calluslines were selected in this example. The SC716 callus line was recoveredfrom an electroporation at 140 μf, 100 V. Three AT824 callus lines wererecovered from 140 μf, 70 V electroporations and four AT824 callus lineswere recovered from electroporation at 140 μf, 140 V.

Three bialaphos resistant callus lines selected in this example, onederived from SC716 and two derived from AT824, were randomly chosen andassayed for phosphinothricin acetyltransferase (PAT) activity. PAT isthe bar gene product, and PAT activity is determined by the ability oftotal protein extracts from potentially transformed cells to acetylatephosphinothricin (PPT), using ¹⁴ C-acetyl coenzyme A as the acetyldonor. This transfer is detected, using thin layer chromatography andautoradiography, by a shift in the mobility of ¹⁴ C labelled compoundfrom that expected for ¹⁴ C-acetyl coenzyme A to that expected for ¹⁴C-N-acetyl PPT. The assay used for detection of PAT activity has beendescribed in detail (Adams et al., published PCT application no.W091/02071; Spencer et al. 1990). All three callus lines testedcontained PAT activity.

In this example, suspension culture cells were electroporated with asecond plasmid, pDPG208, encoding β-glucuronidase (GUS). Detection ofGUS activity can be performed histochemically using5-bromo-4-chloro-3-indolyl glucuronide (X-gluc) as the substrate for theGUS enzyme, yielding a blue precipitate inside of cells containing GUSactivity. This assay has been described in detail (Jefferson 1987). Oneof the seven AT824 callus lines selected in this example, EP413-13,contained cells that turned blue in the histochemical assay. The callusline derived from SC716 in this example did not contain detectable GUSactivity.

Southern blot analysis was performed on three bialaphos resistant calluslines to determine the presence and integration of the bar gene ingenomic callus DNA. Southern blot analysis was performed as follows.Genomic DNA was isolated using a procedure modified from Shure et al.(1983). Approximately one gram of callus tissue from each line waslypholyzed overnight in 15 ml polypropylene tubes. Freeze-dried tissuewas ground to a powder in the tube using a glass rod. Powdered tissuewas mixed thoroughly with 3 ml extraction buffer (7.0 M urea, 0.35 MNaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1% sarcosine). Tissue/bufferhomogenate was extracted with 3 ml phenol/chloroform. The aqueous phasewas separated by centrifugation, and precipitated twice using 1/10volume of 4.4 M ammonium acetate pH 5.2, and an equal volume ofisopropanol. The precipitate was washed with 75% ethanol and resuspendedin 100-500 μl TE (0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Genomic DNAwas digested with a 3-fold excess of restriction enzymes,electrophoresed through 0.8% agarose (FMC), and transferred (Southern,1975) to Nytran (Schleicher and Schuell) using 10× SCP (20× SCP: 2 MNaCl, 0.6 M disodium phosphate, 0.02 M disodium EDTA). Filters wereprehybridized in 6× SCP, 10% dextran sulfate, 2% sarcosine, and 500μg/ml heparin (Chomet et al.,1987) for approximately 10 minutes. Filterswere hybridized overnight at 65° C. in 6× SCP containing 100 μg/mldenatured salmon sperm DNA and ³² P-labelled probe. Probe was generatedby random priming (Feinberg and Vogelstein, 1983); Boehringer-Mannheim).Hybridized filters were washed in 2× SCP, 1% SDS at 65° for 30 minutesand visualized by autoradiography using Kodak XAR5 film.

In this example, genomic DNA isolated from bialaphos resistant calluslines was digested with HindIII and EcoRI, which release a 1.9 kb barfragment from pDPG165 (FIG. 1A). Genomic DNA was probed with ³² Plabelled 0.6 kb SmaI bar fragment from pDPG165 (FIG. 1A). All threeEP413 callus lines analyzed contained DNA that hybridized to the barprobe. Copy number in the transformed callus ranged from one to twocopies (EP413-3) to greater than 20 copies of bar (EP413-1).Furthermore, the restriction digest used, yielded bar-hybridizingfragments in callus DNA samples that were larger than the bar fragmentreleased from pDPG165 in the same restriction digest. This result isindicative of stable integration of introduced DNA into the maizegenome.

Thirty-nine plants were regenerated from seven of the eight bialaphosresistant callus lines selected in this example. Plants were regeneratedfrom six AT824 callus lines and the single SC716 callus line. The plantregenerated from the SC716 callus line (EP413-4) did not survive tomaturity. For plant regeneration, callus growing on 227 mediumcontaining 3 mg/L bialaphos, was transferred to 189 medium (Table 1).Somatic embryos matured on 189 medium after one, two, or three two weeksubculture periods in the dark at 25° C. As somatic embryos developed on189 medium, clumps of tissue containing these embryos were transferredto growth regulator free 101 medium (Table 1) and placed in the light(25-250 μE M⁻² s⁻¹). Plantlets developed on this medium after one, two,or three subculture periods. Plantlets were subsequently transferred to501 medium (Table 1) in Plant Con^(R) containers for rooting and furthergrowth. Regenerates (R₀ plants) were subsequently transferred to asoilless mix in 0.5 liter pots and acclimated to ambient humidity in agrowth chamber (200-450 μE M⁻² s₋₁ ; 14 h photoperiod). The soilless mixhas been described in detail (Adams et al., published PCT applicationno. WO91/02071). Plants were then transferred to a soilless mix in 16liter pots and grown to maturity in a greenhouse.

Plants regenerated from five different EP413 callus lines were assayedfor PAT activity as described for callus earlier in this example. Allfive plants contained PAT activity. Three plants regenerated from thesingle EP413 callus line that exhibited GUS activity (EP413-13) wereanalyzed for GUS activity. All three EP413-13 R₀ plants were positivefor GUS activity. Files of blue cells were observed in leaf tissue ofEP413-13 plants upon incubation with X-Gluc.

EP413 R₀ plants were also analyzed for the presence and integration ofbar by Southern blot. DNA was isolated from leaf tissue as described forcallus except that fresh, rather than lypholyzed tissue was used. Priorto the addition of extraction buffer, fresh leaf tissue was frozen inliquid nitrogen and ground to a fine powder in a 15 ml polypropylenetube using liquid nitrogen and a glass rod. DNA was isolated from fourEP413 R₀ plants, each representing a different callus line. DNA wasanalyzed, digested with HindIII and BglII, or undigested, forhybridization to bar. R₀ DNA was probed with ³² P labelled 0.6 kb SmaIbar fragment from pDPG165 (FIG. 1A). HindIII/BglII digestion of pDPG165releases a fragment containing 35S-bar of approximately 1.3 kb (FIG.1A). Genomic DNA from all four plants contained at least one copy of thethe 1.3 kb HindIII/BglII 35S-bar fragment. In addition, undigestedgenomic DNA from all four plants exhibited hybridization to bar only inhigh molecular weight DNA (>20 kb), indicating integration of pDPG165into maize chromosomal DNA.

Progeny were recovered from outcrosses made betweenelectroporation-derived, transgenic R₀ plants and non-transformed inbredplants. Four EP413-3 R₀ plants were the first of the plants to reachmaturity and flower. One of the plants was outcrossed as the male to aCD inbred plant. This cross resulted in 22 kernels. Sixteen of thesekernels were planted in soilless mix and all germinated. Approximatelytwo weeks post-germination, the progeny (R₁) plants were analyzed forPAT activity. Three of sixteen plants contained PAT activity.

Four transgenic EP413-3 R₀ plants were also outcrossed as the female,using pollen collected from nontransformed inbred plants. Kernelsdeveloped on ears of all four EP413-3 R₀ plants. Thirty-seven kernelswere recovered from an ear on an EP413-3 R₀ plant treated with pollencollected from a seed-derived, non-transformed FBLL inbred plant.Sixteen of these kernels were planted in soil and twelve germinated.

Eight of these plants were analyzed for PAT activity; three of eightwere positive for PAT activity.

These eight plants were also analyzed by Southern blot hybridization forthe presence of bar. Genomic DNA isolated from these eight EP413-3 R₁plants was digested with restriction enzymes HindIII and BglII, whichrelease a 1.3 kb fragment containing bar from pDPG165 (FIG. 1A). Inaddition to DNA from from the eight EP413-3 R₁ plants, DNA isolated fromEP413-3 callus and DNA from the EP413-3 R₀ plant yielding these eight R₁plants was included in the analysis. Genomic DNA was probed with ³² Plabelled 0.6 kb SmaI bar fragment from pDPG165 (FIG. 1A). Hybridizationto bar was detected in the DNA isolated from callus, R₀ and the three R₁plants that contained PAT activity. Each of the bar-positive plantscontained the expected 1.3 kb HindIII/BglII fragment from pDPG165 (FIG.1A) as well as an additional, larger bar-hybridizing fragment ofapproximately 2.0 kb. This result, as well as the PAT activity found tobe present in EP413-3 R₁ plants, conclusively demonstrates the sexualtransmission to progeny of a functional gene introduced into maize cellsby electroporation.

EXAMPLE 24 Transformation of H99 Immature Embryos Using Bialaphos as theSelective Agent Following Electroporation

Immature embryos of H99 were electroporated as described in example 14.Five days after electroporation embryos were transferred to Gelgrosolidified 726 medium containing 0.2 M mannitol. Two days later embryoswere transferred to selection medium, 726 medium containing 1 mg/lbialaphos, 16 embryos per dish.

Embryos were cultured in the dark at 24° C. for about seven weeks.Seventy-eight of the approximately one hundred and twenty embryos platedon selection medium produced Type I callus. All responding callus wastransferred to modified MS-based medium containing 1 mg/l NAA, 1 mg/lBAP and 3% sucrose solidified with 8 g/l Bactoagar (20 medium) formaturation. After about two weeks the tissue was transferred to 20medium containing 1 mg/l bialaphos. Two weeks later the tissue wastransferred to a modified MS-based medium containing 0.5 mg/l NAA, 0.5mg/l BAP and 2% sucrose solidified with 8 g/l Bactoagar (7 medium) with1 mg/l bialaphos. For rooting the tissue was transferred to a modifiedMS-based medium containing 0.25 mg/l NAA, 0.25 mg/l BAP and 2% sucrosesolidified with 8 g/l Bactoagar and finally 1/2 strength MS0.

Tissue from twenty-three of initial seventy-eight responding embryossurvived the regeneration and selection and produced plants. A total ofseventy-five plants were transferred to soil. Four of the plants diedand the remaining seventy-one plants were transferred to the greenhouse.

Plants in the greenhouse were tested for the presence of the bar gene byPCR analysis of DNA extracted from leaf tissue or by painting the leaveswith 2% Basta. One PCR positive plant from embryo # 31 was identified.All of the other plants were either PCR negative or showed severenecrosis in the Basta painting assay. DNA from leaf tissue of plant 3101was further analyzed by Southern blot hybridization and gave a positivesignal when probed with the Sma I fragment of pDPG165. This plant wasselfed on June 7 and June 8 and backcrossed on Jun. 9, 1993. Nine seedwere harvested from this plant. All seed germinated and seven of nine R₁plants contain the bar gene as determined by PCR analysis.

EXAMPLE 25 Transformation of AB12 Using Hygromycin as a Selective AgentFollowing Particle Bombardment

AB12 callus was bombarded as described in example 9. Callus wastransferred (ten 25 mg clumps per plate) onto 734 medium (see Table 1)containing 15 mg/l hygromycin B (Calbiochem) immediately afterbombardment. After 14 days all tissue was transferred to round 2selection plates that contained 60 mg/l hygromycin. After 21 days on theround 2 selection plates, most of the material was transferred to Fmedium containing 60 mg/l hygromycin (round 3 selection plates). Bothround 2 and round 3 plates were then observed periodically for theappearance of viable sectors of callus. Putative transformed callus linePH1 was observed on a round 2 plate, 70 days after bombardment. Putativetransformants PH2 and PH3 were observed on round 3 plates, 58 and 79days after bombardment, respectively. Lines were then maintained on Fmedium containing 60 mg/l hygromycin. Plant regeneration and analysis oftransformants are described in Walters et al. (1992). Fertile transgenicplants were regenerated and transmission of the chimeric gene forhygromycin resistance was demonstrated through two complete generations.

EXAMPLE 26 Transformation of AT824 Using Glyphosate as A Selective AgentFollowing Particle Bombardment

A mutant maize EPSPS gene was introduced into AT824 suspension culturecells via particle bombardment as described in example 10. In thisexample, the mutant maize EPSPS gene was carried by plasmid pDPG436.Plasmid pDPG436 contains a maize EPSPS gene with two amino acid changes,Thr to Ile at position 102 and Pro to Ser at position 106. In thisplasmid, the mutant maize EPSPS expression cassette contains a 35Spromoter/adh1 intron I combination and the nos 3' end. Followingbombardment with gold particles coated with pDPG436, AT824 cells werecultured on 279 medium (Table 1) for four days. Subsequently, the cellswere returned to liquid 401 medium (Table 1), at a density of 2 mlpacked cell volume (PCV) per 20 ml, and cultured for four days. Thecells were then transferred, at a density of 2 ml PCV/20 ml, to fresh401 medium containing 1 mM glyphosate and cultured for four days. Thesubculture into 401 plus 1 mM glyphosate was repeated and after fourdays the cells were plated at a density of 0.1 ml PCV per 100×15 mmpetri dish containing 279 plus 1 mM glyphosate. Six to eight weeks afterbombardment, glyphosate resistant colonies were removed from theselection plates and subcultured onto fresh 279 plus 1 mM glyphosate.Seven glyphosate resistant callus lines were recovered in this example,at a frequency of zero to seven callus lines per bombardment. Tworandomly chosen callus lines were analyzed for the presence of theintroduced DNA by Southern blot hybridization (see example 23). GenomicDNA isolated from the callus lines was analyzed undigested or digestedwith NotI, which releases the the mutant EPSPS expression cassette frompDPG436. The callus DNA was probed with ³² P-labelled nos fragment. Thenos fragment was isolated as an approximately 250 bp NotI/XbaI fragmentfrom pDPG425. The nos fragment was chosen as the probe to avoidbackground hybridization possible using an EPSPS probe due to thepresence of an endogenous maize EPSPS gene. Genomic DNA from both calluslines was positive for hybridization to nos in both the undigested andNotI-digested samples. Both callus DNA samples contained nos-hybridizingbands identical in size to the 35S/adh1 intron I-EPSPS-nos fragmentsreleased from pDPG436 upon digestion with NotI, as well as additionalnos-hybridizing bands.

In a second experiment, AT824 cells were bombarded with a mutant EPSPSgene under control of the rice actin promoter and intron (Cao et al.,Plant Cell Rep (1992) 11:586-591). The plasmid used, pDPG434, containsthe rice actin 5' region, the mutant EPSPS gene described in theprevious example, and the nos 3' end . Bombarded AT824 cells werecultured and selected as described in the previous example. Thirteenglyphosate resistant callus lines were isolated in this example. Four toseven glyphosate resistant callus lines were recovered per bombardmentin this example. As in the previous example, two randomly chosen calluslines were analyzed for the introduced DNA by Southern blothybridization. Using the same analysis as in the previous example, bothcallus lines were found to contain DNA sequence that hybridized to thenos probe, confirming introduction of, and selection for expression of,the introduced mutant EPSPS gene.

H. Identification of Transformed Cells Using Screenable Markers

In addition to selectable markers such as the bar and aroA genes,various screenable marker genes have been employed by the presentinventors in maize transformation. It is contemplated that screenablemarkers may be used to ultimately achieve three objectives: (1) thedetection of expressing colonies in a population, which may notnecessarily employ a visible marker; (2) the visualization, bymicroscope or unaided eye, of expressing cells within a population ortissue; and (3) the ability to assess tissue- and/or cell-specificexpression in gene expression studies. A screenable marker which meetseither objective would be useful and one that meets both criteria wouldbe particularly advantageous. Of the potential candidates beingconsidered as screenable markers, luciferase (Example 27) and aequorinsatisfy only the first requirement, while modified extensin (Example 28)and tyrosinase could potentially meet both goals.

Tyrosinase

The tyrosinase gene is considered to be a potential screenable marker.Normally, melanin production requires the expression of two genes whichencode for tyrosinase and a Cu⁺⁺ transfer protein. Recently, an E. colitransformant was isolated by Claudio Denoyo (Pfizer) in which theCu-transfer protein does not appear to be required. There is still arequirement for copper as a coenzyme for tyrosinase, but this issatisfied by 1 mM Cu⁺⁺ and the tyrosinase acts as a copper scavenger.The gene itself is small with a high GC content which should beexpressed in maize.

Aequorin

This gene was cloned by Dr. M. Cormier (University of Georgia) andencodes a protein called apoaequorin that is normally produced injellyfish. When this protein complexes with a class of lipophilicfluorophores referred to as coelenterazines, the activated complexbecomes sensitive to Ca⁺⁺. When the complex comes into contact withCa⁺⁺, the coelenterazine is reduced to an amide and a photon of light isemitted. Thus, this gene encoded the proteinaceous portion of acalcium-sensitive bioluminescent complex.

This gene has been placed behind the 35S promoter and used to generateaequorin expressing tobacco plants which are being employed to studycalcium levels in plant tissue. However, there are certain technicaldifficulties with developing this system into a screenable marker.Firstly, coelenterazines are difficult to obtain. Secondly, theintensity of the bioluminescence emitted by this complex is probably anorder of magnitude lower than luciferase and the detection systemsneeded to visualize this reaction are very sophisticated and expensive.

However light emission from aequorin expressing cells have never beenmeasured while the cells were being flooded with both coelenterazine andCa⁺⁺. This is an important point. The apoaequorin protein is not therate limiting factor in this reaction, it is the regeneration of reducedcoelenterazine from the coelenteramide. Thus there is a requirement fora strong reducing agent in the assay. Using 1% DMSO, coelenterazine andcalcium could drive the light emission up to detectable levels.Conversely, when these substrates are not present, i.e. in the plant,this normally energy-requiring reaction would not be occurring.

EXAMPLE 27 Luciferase as a Screenable Marker

The lux gene, encoding firefly luciferase, was initially tested as apotential screenable marker using C16 protoplast electroporation toevaluate transient expression, and cotransformation of BMS. Using X-rayfilm to detect bioluminescence, transient expression in C16 protoplastswas detected, but expression in BMS was not high enough for detection onX-ray film. Published results on luciferase expression in tobacco (Ow etal., 1986), indicate that as few as 350 expressing cells could be viablydetected.

Two technical developments prompted the inventors to re-examine thefeasibility of using this marker. The first is a Polaroid ASA 20,000ELISA-type film detection system that is easy to use, with sensitivitycomparable to, or slightly more sensitive than, X-ray film. The secondis a new Luciferase Assay System (Promega), which through the oxidationof luciferyl-CoA, as opposed to luciferin, is claimed to provide a lightreaction with greater total intensity and with a greatly extendedhalf-life.

While the scintillation counter and multiwell luminometer afford onemeans of testing the utility of luciferase screening, i.e. populationalscreening for bioluminescence, it would be ideal to be able to visualizetransformants on the tissue culture plate. This would be even morevaluable if the method was not limited to specific cultures and/ortissue types, and if it could be extended to the whole plant (i.e. forgene expression studies). Computer-enhanced video microscopy has beenrecognized as a potentially valuable tool for these applications.Recently, the Photon Counting Camera (Hamamatsu) has provided a newlevel of sensitivity for the video-imaging of bioluminescence.

Bombarded E1 suspension cells were assayed using both the scintillationcounter and the Polaroid detection system. Extracting 1/4 of the cellson a filter 48 hours after bombardment, luciferase activity was at thelower limit of detection using the scintillation counter. Using muchsmaller aliquots of cells, due to the microtiter wells in the assaysystem, no discernible activity was observed using the Polaroiddetection system.

Using dilutions of purified luciferin in both the scintillation counterand the Polaroid system, and comparing these results to scintillationcounts of transient lux expression after bombardment of E1 cells, it wasestimated that there is probably only a 10-fold discrepancy between thetransient expression levels and the ability to detect a signal usingthis film. Further studies were thus conducted to bridge this detectiongap. The luciferase system was optimized both with respect to the assaymixture and also with the creation of further luciferase expressionvectors.

Luciferase Assay Mixture

The first step in this process was to re-evaluate the composition of theassay mixture, both in terms of relative luminescence and subsequentviability of the tissue. The addition of coenzyme A to the reaction mixhas been reported to improve the bioluminescence kinetics of theluciferase assay. This has been one of the features incorporated intothe Luciferase Assay System sold by Promega. In comparing the Promegamixture to the luciferase assay mixture usually employed, no significantdifference was observed in assaying purified luciferase enzyme. However,when cell extract from a transformed E1 callus line was used, thePromega mixture produced approximately an 18-fold increase in signal(measured over a two minute period using the scintillation counter).

Multiple experiments were performed to assess the influence of thecomponents of these assay mixtures; varying both the species andconcentration of reducing agent, salts, coenzymes, protective proteins,and the substrate itself, luciferin. The type and amount of reducingagent was a point of concern for tissue viability, so two alternativeswere compared for signal strength and tissue viability. Both glutathione(i.e. 10-50 mM) and DTT (5-33 mM) were found to produce strongbioluminescence signals in intact cell clusters, although the resultswere more variable with glutathione. 5 mM DDT provided the bestcompromise for enhanced signal strength and growth of callus afterexposure to the mixture for 20 minutes. The most effective combinationfound to date is a hybrid, taking components from both the Promegarecipe and the previously used standard mixture. This combinationresulted in a 2-4 fold increase in signal over the Promega mixture. Therecipe for this improved mix is:

25 mM Tris, PO₄ (pH 7.8), 1% BSA (Fraction 5), 5 mM DTT, 1 mM EDTA, 0.3mM ATP, 8 mM MgCl₂, 0.47 mM firefly luciferin, 0.3 mM coenzyme A.

Detection of Luciferase Expression

Once the assay mixture had been optimized, the scintillation counter andluminometer were evaluated as to their utility for screening oftransformants. Using cell clusters ranging between 100 to 200 um indiameter, both instruments were capable of detecting luciferase activityin transformants with high expression levels. The luminometer was moresensitive, being able to detect transformants with lower expressionlevels and/or smaller groups of cells consistently. Plastic covers forthe multiwell dishes were obtained that have a minimal effect onreducing the bioluminescent signal, and allow this assay to be performedunder sterile conditions.

Reconstruction studies were initiated, placing 10-15 small transformedtissue pieces (all below 150 μm diameter) into 0.75 ml ofnon-transformed suspension cells. Initial screening was encouraging asluciferase activity could be detected even within this largenon-transformed population. This also illustrated the advantages anddisadvantages of the two detection devises; the scintillation counter isconvenient for screening large aliquots of cells, while the luminometeris more sensitive but more labor intensive.

To take advantage of the relative merits of both devises, in the secondreconstruction experiment the scintillation counter and then theluminometer were used for sequential screening. Again, 10-15 transformedcell clusters were mixed into a non-transformed population (1.25 ml ofsuspension) and placed on the shaker for 2 hours. The suspension wasthen pipetted into seven scintillation vials and assayed for luciferaseactivity. Positive signals were recorded for 5/7 samples, and these fivewere pipetted onto fresh 227 solid medium and allowed to grow for oneweek. At this time, two of the samples were aliquoted into multiwelldishes and assayed for activity using the luminometer. Each of thesewells contained approximately 25 μl of tissue. Six and 7 positive wellswere recorded for these two samples, and the tissue again wastransferred back onto fresh 227 medium.

These results exemplify the power of the luminometer, because the samplesize in each well is small the enrichment is much greater (this singlescreen eliminated approximately 95% of the population). The drawback tothis type of screening is the amount of tissue manipulation and risk ofcontamination. Despite some contamination it is extremely encouragingthat the enrichment technique was successful and that the tissueremained healthy (based on visual assessment and subsequent growth).

Improved Luciferase Expression Vectors

Recent improvements in the luciferase assay mixture increased thesensitivity enough so that detection of stable transformed sectorsappeared feasible. To further improve the chances of successfullyscreening for transformants, expression should also be optimized.Towards this goal, two new luciferase vectors were constructed to boostexpression levels in maize cells. Both vectors utilize intron VI fromAdh1 (derived from vector pDPG273) fused to firefly luciferase (obtainedfrom vector pDPG215). These elements were inserted into either thepDPG282 (4 OCS inverted-35S) or the pDPG283 (4 OCS-35S) backbone (barwas excised as a BamHI/Nhe1 fragment and the intron plus luciferase geneinserted). The 4 OCS-35S promoter has been shown with the uidA gene togive very high levels of transient expression. When this promoter isfused to luciferase it was anticipated that it would result in highlevels of expression.

Transient expression levels from each of these vectors was determined inbombarded E1 suspension culture cells. The two new vectors were alsocompared to pDPG215 (35S-intron l-luciferase-Tr7 3') as the standard. Atleast for transient expression, the 4 OCS-35S promoter was not found tobe better than the 35S promoter. Vector pDPG351 gave significantlyhigher levels of transient expression than pDPG350, but not higher thanpDPG215.

For independent stable transformants, a wide range of luciferaeexpression has been observed in experiments using either pDPG215 orpDPG315. For both plasmids luciferase expression as measured in callususing the scintillation counter ranged between 100 and 2×10⁶ CPM.Luciferase expression has been confirmed in R₀ plants.

Microspore-derived cell clusters (genotype G238) were bombarded with thep350 and p351 constructs. This tissue was grown on non-selective 227medium, and was screened 2-3 weeks post-bombardment using the multiwellluminometer. Out of six plates screened, two wells produced readingspotentially above background. This tissue was transferred to fresh 227.Also, AT824 suspension samples were bombarded with the p350 and p351.After 3 days on the filter, the tissue was put back into liquid andscreening for luciferase activity was started 12 days post-bombardment.

EXAMPLE 28 Extensin: A Secreted Screenable Marker

Initially, candidates considered as screenable markers have been genesencoding intracellular proteins that require diffusible substrates orpermeation of cells to perform the assay. An alternative is a secretablemarker. Three general types have been considered: (i) functionalsecreted enzymes detectable by assaying catalytic activity, (ii) smalldiffusible proteins detectable by ELISA, such as IL-2, or (iii) secretedmarkers that remain sequestered in the cell wall that also containunique epitopes for antibody detection.

Candidates for functional secreted markers that could be detectedthrough catalytic action would include such enzymes as β-galactosidaseand β-glucuronidase. Unfortunately, these enzymes are modified duringthe secretion process in a manner which renders them inactive. Forexample, GUS enters the endoplasmic reticulum and is N-glycosylatedwhich blocks enzyme activity (Iturraga et al., 1989). Recently, theN-linked glycosylation site was altered by site-directed mutagenesis(Farrell & Beachy, 1990), but no reports on secretion and/or functionalactivity have yet followed. In using GUS, it is not clear whether thecalorimetric product would remain localized to the point where cleardemarcation of expressing and non-expressing cells could be achieved,but this is still a promising marker.

A number of small proteins such as interleukins have been wellcharacterized in terms of molecular genetics and immunodetection.However, even if properly targeted across the plasma membrane, the bestcandidates are all small enough to diffuse readily through the cell wallinto the extracellular solution. Thus, they could be detected by ELISAmethods, but could not be localized to specific cells. A variety ofmammalian genes are known that would provide a unique epitope forlabeling. However, large mammalian proteins secreted across the plasmamembrane would not be likely to reach the surface of the wall (and hencebe relatively inaccessible), while small proteins would diffuse into theextracellular space.

The requirements of a secreted antigen construct were thus determined tobe: encoding a unique epitope sequence that would provide low backgroundin plant tissue; a promoter-leader sequence that would impart efficientexpression and targeting across the plasma membrane; and the productionof a protein which is bound in the cell wall and yet accessible toantibodies. The expression of a modified, but otherwisenormally-secreted, cell wall constituent was considered to be an idealcandidate for a secretable marker. Extensin, HPRG, was the cell wallprotein chosen since this molecule is fairly well characterized in termsof molecular biology, expression and protein structure, and the maizegenomic HPRG sequence was available (from Dr. Pedro Puigdomenech).

The strategy for visualizing the expression of the introduced extensingene revolves around introducing a novel epitope into the secretedprotein, which could then be localized using immunological techniques.Certain preliminary tests need to be performed before making such aconstruct. A novel epitope must be identified for which a high-titerantibody is available; maize extracts should be reacted with theantibody to ensure there is no non-specific background labeling; and thefeasibility of immunolabeling the cell wall of living cells should bedetermined.

The epitope chosen was a 15 amino acid sequence from the pro-region ofmurine interleukin-1-β, MATVPELNCEMPPSD (seq id no:1), which wasrecognized by polyclonal antibodies. A dot-blot was performed loadingeither 0, 10, or 50 ng of a 31 kd recombinant protein, and testing witheither R1682 serum, normal rabbit serum, Fc-purified R1684, orFc-purified normal rabbit. With the higher protein load (50 ng),background was observed in the normal rabbit and the Fc-purified normalrabbit at the higher antibody concentrations (i.e. 1:300, and 1:100). Nobackground was observed in the 10 ng dots at antibody dilutions greater(less concentrated) than 1:100. For the R1682 serum, protein wasdetected for both the 10 and 50 ng dots at all antibody dilutions, evendown to 1:3000. The Fc-purified R1684 was approximately 10-fold lesssensitive. This result indicates that the R1682 antibody is veryhigh-titer, and should be useful as a marker system.

On analyzing the IL-1-β pro sequence in computer gene and protein databanks, no sequence homology was found in either plants or fungi. Testingextracts from maize suspension cells and from cell walls confirmed thatno background labeling exists.

Using colloidal gold conjugated secondary antibody followed by silverenhancement, surface labeling of "living" root tips was verified. Thelabeling of the surface with both the primary and secondary antibodieswas performed under physiological conditions (low concentrations oforganic buffers and/or salts). However, in order to visualize the goldlabel a silver enhancement process was then utilized, and this is toxic.The use of a new fluorophore, phycoerythrin, conjugated to the secondaryantibody is also contemplated. This should eliminate problems withendogenous background fluorescence and increase resolution.

The necessary oligonucleotides were made and a cloning strategy wasdeveloped for inserting the novel IL-1 sequence into the carboxyl-end ofthe extensin structural gene and placing this into a plant expressionvector (CaMV 35S promoter, Agrobacterium tumefaceiens transcipt 7 3'region).

K. Co-Transformation

Co-transformation may be achieved using a vector containing the markerand another gene or genes of interest. Alternatively, different vectors,e.g., plasmids, may contain the different genes of interest, and theplasmids may be concurrently delivered to the recipient cells. Usingthis method, the assumption is made that a certain percentage of cellsin which the marker has been introduced, have also received the othergene(s) of interest. As can be seen in the following examples, not allcells selected by means of the marker, will express the other genes ofinterest which had been presented to the cells concurrently. Forinstance, in Example 29, successful cotransformation occurred in 17/20independent transformants (see Table 5), coexpression occurred in 4/20.In some transformants, there was variable expression among transformedcells.

EXAMPLE 29 Co-Integration and Co-Expression of the Bar Gene and the GUSGene to Cell Lines Derived from the SC82 Suspension Culture

Of the bialaphos-resistant isolates selected from a reinitiation ofcryopreserved SC82 cells transformed with separate plasmids (asdescribed for SC716), nineteen independent transformants were selectedin this experiment (experiment #6, Table 5). The frequency ofcointegration and coexpression in those isolates was similar to thatdescribed for SC716 isolates (Table 5). The pattern of GUS staining inthese transformants varied in a manner similar to that described forcoexpressing SC716 transformants. A transformant, Y13, which containedintact GUS coding sequence, exhibited varying levels of GUS activity asshown in FIG. 6. This type of expression pattern has been describedpreviously in cotransformed BMS cells (Klein et al., 1989). Variableactivity detected in the cells from a single transformant may beattributed to unequal penetration of the GUS substrate, or differentialexpression, methylation, or the absence of the gene in some cells.

These results show that both the bar gene and the GUS gene are presentin some of the cells bombarded with the two plasmids containing thesegenes. Co-transformation has occurred. In the cotransformation examplesdescribed herein and summarized in Table 5, cotransformation frequencyof the non-selected gene was 77%; coexpression frequency was 18%.

L. Regeneration of Plants From Transformed Cells

For use in agriculture, transformation of cells in vitro is only onestep toward commercial utilization of these new methods. Plants must beregenerated from the transformed cells, and the regenerated plants mustbe developed into full plants capable of growing crops in open fields.For this purpose, fertile corn plants are required. The inventiondisclosed herein is the first successful production of fertile maizeplants (e.g., see FIG. 7A) from transformed cells.

During suspension culture development, small cell aggregates (10-100cells) are formed, apparently from larger cell clusters, giving theculture a dispersed appearance. Upon plating these cells to solid media,somatic embryo development can be induced, and these embryos can bematured, germinated and grown into fertile seed-bearing plants. Thecharacteristics of embryogenicity, regenerability, and plant fertilityare gradually lost as a function of time in suspension culture.Cryopreservation of suspension cells arrests development of the cultureand prevents loss of these characteristics during the cryopreservationperiod.

EXAMPLE 30 Regeneration of Plants from SC82 and SC716

One efficient regeneration system involves transfer of embryogeniccallus to MS (Murashige & Skoog, 1962) medium containing 0.25 mg/l2,4-dichlorophenoxyacetic acid and 10.0 mg/l 6-benzyl-aminopurine.Tissue was maintained on this medium for approximately 2 weeks andsubsequently transferred to MS medium without growth regulators(Shillito et al., 1989). Shoots that developed after 2-4 weeks on growthregulator-free medium were transferred to MS medium containing 1%sucrose and solidified with 2 g/l Gelgro® in Plant Con® containers whererooting occurred.

Another successful regeneration scheme involved transfer of embryogeniccallus to N6 (Chu et al., 1975) medium containing 6% sucrose and nogrowth regulators (Armstrong & Green, 1985) for two weeks followed bytransfer to MS medium without growth regulators as described above.Regeneration was performed at 25° C. under fluorescent lights (250microeinsteins·m⁻² ·s⁻¹). After approximately 2 weeks developingplantlets were transferred to a Plant Con® container containing medium501. When plantlets had developed 3 leaves and 2-3 roots they weretransferred to soil, hardened off in a growth chamber (85% relativehumidity, 600 ppm CO₂, 250 microeinsteins·m⁻² ·s⁻¹), and grown tomaturity either in a growth chamber or the greenhouse.

Regeneration of plants from transformed cells requires careful attentionto details of tissue culture techniques. One of the major factors is thechoice of tissue culture media. There are many media which will supportgrowth of plant cells in suspension cultures, but some media give bettergrowth than others at different stages of development. Moreover,different cell lines respond to specific media in different ways. Afurther complication is that treatment of cells from callus initiationthrough transformation and ultimately to the greenhouse as plants,requires a multivariate approach. A progression consisting of variousmedia types, representing sequential use of different media, is neededto optimize the proportion of transformed plants that result from eachcell line. Table 6 illustrates one sequential application ofcombinations of tissue culture media to cells at different stages ofdevelopment. Successful progress is ascertained by the total number ofplants regenerated.

It can be seen that using the same group of media, cell lines will varyin their success rates (number of plants) (Table 6). There was alsovariation in overall success rate, line AO1-15 yielding the greatestnumber of plants overall. (It should be noted, however, that becausetissue was limiting not all combinations of media were used on alllines, therefore, overall comparisons are limited.)

A preferred embodiment for use on cell lines SC82 and SC716, at leastinitially, is the combination shown in the second column under theregeneration media progression (media 227, 171, 101, 501). Media 227 isa good media for the selective part of the experiments, for example, touse for growth of callus in the presence of bialaphos. This mediacontains the growth regulator dicamba. NAA and 2,4-D are growthregulators in other media. In liquid media, these may be encapsulatedfor controlled release (Adams, W. et al., in preparation).

Thus, it can be seen from Table 1 that the various media are modified soas to make them particularly applicable to the development of thetransformed plant at the various stages of the transformation process.For example, subculture of cells in media 171 after applying theselective agent, yields very small embryos. Moreover, it is believedthat the presence of BAP in the media facilitates development of shoots.Myo-inositol is believed to be useful in cell wall synthesis. Shootelongation and root development proceeds after transfer to media 101.101 and 501 do not contain the growth regulators that are required forearlier stages of regeneration.

                                      TABLE 6                                     __________________________________________________________________________    Plants to Soil From Bombardment of SC716 (Expts 1, 2; Table 6).                        REGENERATION MEDIA PROGRESSIONS                                                     227b                                                                             227b                                                                             227b                                                                             227b  227b                                                                             227b                                                                             227b  227b                                                                             #                                    227b 201b 52 163 205 227b 201b 205 163 227b 201b PLANTS                      227b 171 171 171 171 171 173 173 173 173 177 177 TO                          Cell Line 101 101 101 101 101 101 101 101 101 101 101 101 SOIL              __________________________________________________________________________    CONTROLS                                                                        A01C-11 X 4 X X X X 2 X X X X X  6*                                           A01C-01 X 7 X X X X 27 X X X X X  34*                                         TOTAL X 11 X X X X 29 X X X X X  40*                                          TRANSFORMED                                                                   A01C-11 X X X 0 0 0 X X 0 0 X X  0                                            A01C-12 X 2 X 0 0 0 X X 0 0 X X  2                                            A01C-13 X 5 1 4 0 0 1 1 1 1 X X  14*                                          A01C-14 X 2 X 0 0 0 X X 1 0 X X  3*                                           A01C-15 X 28 0 12 7 1 23 13 0 0 0 0  84*                                      A01C-17 X 7 0 0 0 0 17 0 0 0 0 0  24                                          A01C-18 X 12 0 0 X 0 21 10 0 X 2 0  45*                                       A01C-19 X 0 X X 0 X 0 X X 0 X 0  0                                            A01C-20 X 10 X 0 0 X 0 X X 0 X 0  10*                                         A01C-21 X 0 X X X X 0 X X X X 0  0                                            A01C-24 2 4 0 0 0 0 6 5 0 0 0 0  17*                                          A01C-25 X 9 X X 0 0 1 X 0 0 X X  10                                           A01C-27 X 0 X X X X 10 X X X X 0  10*                                         TOTAL 2 79 1 16 7 1 79 29 2 1 2 0 219*                                        COMBINED                                                                      CONTROLS X 11 X X X X 29 X X X X X  40*                                       TRANSFORMED 2 79 1 16 7 1 79 29 2 1 2 0 219*                                  TOTAL 2 90 1 16 7 1 108 29 2 1 2 0 259*                                     __________________________________________________________________________     X = Regeneration not attempted by this route.                                 *More plants could have been taken to soil.                                   201b = 201 with 1 mg/l bialophos.                                             2227b = 227 with 1 mg/l bialophos.                                       

Transfer of regenerating plants is preferably completed in anagar-solidified media adapted from a nutrient solution developed byClark (1982), media 501. The composition of this media facilitates thehardening of the developing plants so that they can be transferred tothe greenhouse for final growth as a plant. The salt concentration ofthis media is significantly different from that of the three media usedin the earlier stages, forcing the plant to develop its own metabolicpathways. These steps toward independent growth are required beforeplants can be transferred from tissue culture vessels (e.g. petridishes, plant cans) to the greenhouse.

Approximately 50% of transformed callus lines derived from the initialSC82 and SC716 experiments were regenerable by the routes tested.Transgenic plants were regenerated from four of seven independent SC82transformants and ten of twenty independent SC716 transformants.Regeneration of thirteen independently, transformed cell lines and twocontrol lines of SC716 was pursued. Regeneration was successful from tenof thirteen transformants. Although a total of 458 plantlets wereregenerated, due to time and space constraints only 219 transformedplants (representing approximately 48% of the total number ofregenerants) were transferred to a soilless mix (see below).Approximately 185 plants survived. Twelve regeneration protocols wereinvestigated and the number of plants regenerated from each route hasbeen quantified (Table 6). There appeared to be no significant advantageto maturing the tissues on 201, 52, 163, or 205 (see Table 1 for mediacodes) prior to transfer to medium 171 or 173. The majority of theplants were generated by subculturing embryogenic callus directly from227 to either 171 or 173. These plantlets developed roots withoutaddition of exogenous auxins, and plantlets were then transferred to asoilless mix, as was necessary for many of the transformants regeneratedfrom SC82.

The soilless mix employed comprised Pro Mix, Micromax, Osmocote 14-14-14and vermiculite. Pro Mix is a commercial product used to increasefertility and porosity as well as reduce the weight of the mixture. Thisis the bulk material in the mixture. Osmocote is another commercialproduct that is a slow release fertilizer with anitrogen-phosphorus-potassium ratio of 14:14:14. Micromax is anothercommercial fertilizer that contains all of the essential micronutrients.The ratio used to prepare the soilless mix was: 3 bales (3 ft³ each) ProMix; 10 gallons (vol.) vermiculite; 7 pounds Osmocote; 46 ml Micromax.The soilless mix may be supplemented with one or two applications ofsoluble Fe to reduce interveinal chlorosis during early seedling andplant growth.

Regeneration of transformed SC82 selected cell lines yielded 76 plantstransferred to the soilless mix, and 73 survived. The plants wereregenerated from six bialaphos-resistant isolates, representing four ofseven clonally independent transformants. Eighteen protocols were usedsuccessfully to regenerate the seventy six plants (Table 7). Differencesin morphology between cell lines deemed some protocols more suitablethan others for regeneration.

                                      TABLE 7                                     __________________________________________________________________________    EFFECTS OF PROGRESSION OF MEDIA ON THE NUMBER OF PLANTS REGENERATED           (SC82)*                                                                                                      227B  227B  227B  227B  227B                           227B 227B 227B 227B 227B 227B 227B 201B 227B 201B 201B 201B 201B                                                                  227B 227B                                                                   227B 227A 227A                                                                227A 171 52 52                                                                52 171 201B                                                                   227B 205 227B                                                                 205 1 52                                                                        142 173 171                                                                 205 209 173 173                                                               173 173 171 173                                                               173 178 171 177                                                               177 178 171                                                                    CELL 101 101                                                                 101 101 101 101                                                               101 101 101 101                                                               101 101 101 101                                                               101 101 101 101                                                               # OF                  LINE 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501 501                                                                  501 501             __________________________________________________________________________                                                              PLANTS              B3-14-4                                                                           1  X  14 X  X  X  1  1  X  2  X  X  5  X  5  X  X  X  29                    B3-14-9 X X 1 1 X 4 1 X X X X X X 1 X 1 X X 9                                 B3-14-7 X X X X X X X X X X 6 2 X X X X X 1 9                                 B3-14-6 X X X X 1 X X X X X X X X X X X X X 1                                 B3-14-3 X X X X X X X X X X X X X X X X X X 0                                 B3-14-2 X X X X X X X X X X X X X X X X X X 0                                 B3-14-1 X X X X X X X X X X X X X X X X X X 0                                 B3-14-5 X X X X X X X X X X X X X X X X X X 0                                 B3-13-5 X X X X X X X X X X X X X X X X X X 0                                 B3-13-2 X 1 13 X X X 3 2 2 X X X X X 1 X X X 22                               B3-13-1 X 3 X 1 X X X X 1 X X X X X X X 1 X 6                                 TOTAL 1 4 28 2 1 4 5 3 3 2 6 2 5 1 6 1 1 1 76                               __________________________________________________________________________     *See table 1 for media codes.                                                 X = This media progression was either attempted and unsuccessful or not       attempted.                                                                    227A = 227 with 10.sup.-7 M ABA.                                              227B = 227 with 1 mg/l bialaphos.   }{2060                               

Prior to regeneration, the callus was transferred to either a) anN6-based medium containing either dicamba or 2,4-D or b) an MS-basedmedium containing 2,4-D. These steps allowed further embryoiddevelopment prior to maturation. Most of the maturation media containedhigh BAP levels (5-10 mg/l) to enhance shoot development and causeproliferation. An MS-based medium with low 2,4-D (0.25 mg/l) and highBAP (10 mg/l), as described by Shillito et al., 1989, was found to bequite effective for regeneration.

Likewise, an MS-based medium containing 1 μm NAA, 1 μm IAA, 2 μm 2-IP,and 5 mg/l BAP (modified from Conger et al., 1987) also promoted plantregeneration of these transformants. After plantlets recovered by any ofthe regenerative protocols had grown to five cm, they were transferredto a nutrient solution described by Clark, 1982, supplemented with 2%sucrose and solidified with Gelgro. Plantlets which were slow to developroots were treated with 3 μl droplets of 0.3% IBA at the base of theshoot to stimulate rooting. Plants with well developed root systems weretransferred to a soilless mix and grown in controlled environmentalchambers from 5-10 days, prior to transfer to the greenhouse.

EXAMPLE 31 Regeneration of AT824 Transformants

Transformants were produced as described in examples 19 and 20. Forregeneration tissue was first transferred to solid medium 223 andincubated for two weeks. Transformants may be inititally subcultured onany solid culture that supports callus growth, e.g., 223, 425, 409 andso forth. Subsequently transformants were subcultured one to threetimes, but usually twice on 189 medium (first passage in the dark andsecond passage in low light) and once or twice on 101 medium in petridishes before being transferred to 607 medium in Plant Cons©. Variationsin the regeneration protocol are normal based on the progress of plantregeneration. Hence some of the transformants were first subculturedonce on 425 medium, twice on 189 medium, once or twice on 101 mediumfollowed by transfer to 501 medium in Plant Cons©. As shoots developedon 101 medium, the light intensity was increased by slowly adjusting thedistance of the plates from the light source located overhead. Allsubculture intervals were for about 2 weeks at 24° C. Transformants thatdeveloped 3 shoots and 2-3 roots were transferred to soil.

Plantlets in soil were incubated in an illuminated growth chamber andconditions were slowly adjusted to adapt or condition the plantlets tothe drier and more illuminated conditions of the greenhouse. Afteradaptation/conditioning in the growth chamber, plants were transplantedindividually to 5 gallon pots of soil in the greenhouse.

M. Assays for Integration of Exogenous

DNA and Expression of DNA in R₀ R₁ Plants

Studies were undertaken to determine the expression of the transformedgene(s) in transgenic R₀ and R₁ plants. Functional activity of PAT wasassessed by localized application of a commercial herbicide formulationcontaining PPT to leaves of SC82 R₀ and R₁ plants. No necrosis wasobserved on leaves of R₀ plants containing either high levels (E2/E5),or low levels (E3/E4) of PAT. Herbicide-treated E3/E4/E6 and controlleaves are shown in FIG. 8A. Herbicide was also applied to leaves ofE2/E5 progeny segregating for bar. As demonstrated in FIG. 8B, leaves ofR₁ plants expressing bar exhibited no necrosis six days afterapplication of the herbicide while R₁ plants without bar developednecrotic lesions. No necrosis was observed on transformed leaves up to30 days post-application.

Twenty-one R₀ plants, representing each of the four regenerabletransformed SC82 callus lines, were also analyzed for expression of thebar gene product, PAT, by thin-layer chromatographic techniques. Proteinextracts from the leaves of the plants were tested. PAT activity of oneplant regenerated from each callus line is shown in FIG. 9.

All 21 plants tested contained PAT activity. Furthermore, activitylevels were comparable to levels in the callus lines from which theplants were regenerated. The nontransformed plant showed no PAT activity(no band is in the expected position for acetylated PPT in theautoradiograph from the PAT chromatogram). A band appears in the BMSlane that is not in lanes containing protein extracts from the plantleaves. This extra band was believed to be an artifact.

As another method of confirming that genes had been delivered to cellsand integrated, genomic (chromosomal) DNA was isolated from anontransformed plant, the four regenerable callus lines and from two R₀plants derived from each callus line. FIG. 10 illustrates results of gelblot analysis of genomic DNA from the four transformed calli (C) and theR₀ plants derived from them. The transformed callus and all plantsregenerated from transformed callus contained sequences that hybridizedto the bar probe, indicating the presence of DNA sequences that werecomplementary to bar. Furthermore, in all instances, hybridizationpatterns observed in plant DNA were identical in pattern and intensityto the hybridization profiles of the corresponding callus DNA.

DNA from E3/E4/E6 callus and the desired R₀ plants containedapproximately twenty intact copies of the 1.9 kb bar expression unit(Cauliflower Mosaic Virus 35S promoter-bar-Agrobacterium transcript 73'-end) as well as numerous other bar-hybridizing fragments. E11 callusand plant DNA contained 1-2 copies of the intact expression unit and 5-6additional non-intact hybridizing fragments. E10 callus and plantscontained 1-2 copies of the intact bar expression unit. E2/E5 DNAcontained a single fragment of approximately 3 kb that hybridized to theprobe. To confirm that the hybridizing sequence observed in all plantswere integrated into the chromosomal DNA, undigested genomic DNA fromone plant derived from each independent transformant was analyzed by DNAgel blot hybridization. Hybridization to bar was observed only in highmolecular weight DNA providing evidence for the integration of bar intothe maize genome.

Plants were regenerated from the coexpressing callus line, Y13, shown inFIG. 6. Plants regenerated from Y13 (experiment #6, Table 5) wereassayed for GUS activity and histochemically stained leaf tissue fromone plant is shown in FIGS. 8C, D, E. Numerous cell types includingepidermal, guard, mesophyll and bundle sheath cells stained positive forGUS activity. Staining intensity was greatest in the vascular bundles.Although all leaf samples from the regenerated plants tested (5/5)expressed the nonselected gene, some non-expressing leaf sectors werealso observed. Leaf tissue extracts from three Y13 and three controlplants were also assayed for GUS activity by fluorometric analysis(Jefferson, 1987). Activity detected in two opposing leaves from each ofthree Y13 plants tested was at least 100-fold higher than that incontrol leaves.

EXAMPLE 32 General Methods for Assays

A method to detect the presence of phosphinothricin acetyl transferase(PAT) activity is to use an in vitro enzyme reaction followed by thinlayer chromatography.

An example of such detection is shown in FIG. 9 wherein various proteinextracts prepared from homogenates of potentially transformed cells, andfrom control cells that have neither been transformed nor exposed tobialaphos selection, are assayed by incubation with PPT and ¹⁴ C-AcetylCoenzyme A followed by thin layer chromatography. 25 μg of proteinextract were loaded per lane. The source in lanes E1-E11 were SC82transformants; B13 is a BMS (Black Mexican Sweet corn nonembryogenic)bar transformant. E0 is a nonselected, nontransformed control.

As can be seen at the position indicated by the arrow (the positionexpected for the mobility of ¹⁴ C-N-AcPPT), all lanes except thenontransformed control exhibit PAT activity by the formation of acompound with the appropriate mobility expected for ¹⁴ C-N-Acetyl PPT.Variation in activity levels among the transformants was approximately10 fold, as demonstrated by the relative intensity of the bands. Theresults of this assay provide confirmation of the expression of the bargene which codes for PAT. For analysis of PAT activity in plant tissue,100-200 mg of leaf tissue was extracted in sintered glass homogenizersand assayed as described previously.

GUS activity was assessed histochemically as described using5-bromo-4-chloro-3-indolyl glucuronide (Jefferson, 1987); tissue wasscored for blue cells 18-24 h after addition of substrate. Fluorometricanalysis was performed as described by Jefferson (1987) using 4-methylumbelliferyl glucuronide.

DNA analysis was performed as follows. Genomic DNA was isolated using aprocedure modified from Shure, et al., 1983. Approximately 1 gm callustissue was ground to a fine powder in liquid N2 using a mortar andpestle. Powdered tissue was mixed thoroughly with 4 ml extraction buffer(7.0 M urea, 0.35 M NaCl, 0.05 M Tris-HCl pH 8.0, 0.01 M EDTA, 1%sarcosine). Tissue/buffer homogenate was extracted with 4 mlphenol/chloroform. The aqueous phase was separated by centrifugation,passed through Miracloth, and precipitated twice using 1/10 volume of4.4 M ammonium acetate, pH 5.2 and an equal volume of isopropanol. Theprecipitate was washed with 70% ethanol and resuspended in 200-500 μl TE(0.01 M Tris-HCl, 0.001 M EDTA, pH 8.0). Plant tissue may also beemployed for the isolation of DNA using the foregoing procedure.

The presence of a gene in a transformed cell may be detected through theuse of polymerase chain reaction (PCR). Using this technique specificfragments of DNA can be amplified and detected following agarose gelelectrophoresis. For example the bar gene may be detected using PCR. Twohundred to 1000 ng genomic DNA is added to a reaction mix containing 10mM Tris-HCl pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.1 mg/ml gelatin, 200 uMeach DATP, dCTP, dGTP, dTTP, 0.5 uM each forward and reverse DNAprimers, 20% glycerol, and 2.5 units Taq DNA polymerase. The forwardprimer is CATCGAGACAAGCACGGTCAACTTC (seq id no:16). The reverse primeris AAGTCCCTGGAGGCACAGGGCTTCAAGA (seq id no:17). PCR amplification of barusing these primers requires the presence of glycerol, but thiscomponent is not needed for most other applications. The reaction is runin a thermal cycling machine as follows: 3 minutes at 94° C., 39 repeatsof the cycle 1 minute at 94° C., 1 minute at 50° C., 30 seconds at 72°C., followed by 5 minutes at 72° C. Twenty ul of each reaction mix isrun on a 3.5% NuSieve gel in TBE buffer (90 mM Tris-borate, 2 mM EDTA)at 50 V for two to four hours. Using these primers a 279 base pairfragment of the bar gene is amplified.

For Southern blot analysis genomic DNA was digested with a 3-fold excessof restriction enzymes, electrophoresed through 0.8% agarose (FMC), andtransferred (Southern, 1975) to Nytran (Schleicher and Schuell) using10× SCP (20× SCP: 2 M NaCl, 0.6 M disodium phosphate, 0.02 M disodiumEDTA). Filters were prehybridized at 65° C. in 6× SCP, 10% dextransulfate, 2% sarcosine, and 500 μg/ml heparin (Chomet et al., 1987) for15 min. Filters were hybridized overnight at 65° C. in 6× SCP containing100 μg/ml denatured salmon sperm DNA and ³² P-labeled probe. The 0.6 kbSmaI fragment from pDPG165 and the 1.8 kb BamHI/EcoRI fragment frompCEV5 were used in random priming reactions (Feinberg & Vogelstein,1983; Boehringer-Mannheim) to generate labeled probes for detectingsequences encoding PAT or GUS, respectively. Filters were washed in 2×SCP, 1% SDS at 65° C. for 30 min. and visualized by autoradiographyusing Kodak XAR5 film. Prior to rehybridization with a second probe, thefilters were boiled for 10 min. in distilled H₂ O to remove the firstprobe and then prehybridized as described above.

N. Fertility of Transgenic Plants

To recover progeny the regenerated, genetically transformed maize plants(designated R₀), were backcrossed with pollen collected fromnontransformed plants derived from seeds. Alternatively pollen wascollected from R₀ plants and used to pollinate nontransformed plants.Progeny (designated R₁) that contained and expressed bar were recoveredfrom crosses in which the transformant was used as a male or femaleparent.

An important aspect of this invention is the production for the firsttime of fertile, genetically transformed maize plants (R₀) and progeny(R₁). These were regenerated from embryogenic cells that weretransformed. R₁ plants are those resulting from backcrossing of R₀plants.

Pollination of transgenic R₀ ears with non-transformed B73 pollenresulted in kernel development. In addition, kernels developed frompistillate flowers on male inflorescences that were pollinated withnon-transformed B73 pollen. Kernels on transformed R₀ plants from SC82developed normally for approximately 10-14 days post-pollination butafter this period the kernels ceased development and often collapsed.Most plants exhibited premature senescence at this time. A total of 153kernels developed sporadically on numerous plants (see Table 8): 8 of 37E2/E5 plants, 2 of 22 E10 plants, and 3 of 6 E11 plants. Viable progenywere recovered by embryo rescue from 11 E2/E5 plants and one E10 plant.

SC716 R₀ plants were also backcrossed with seed-derived B73 plants. Todate, from the 35 mature SC716 R₀ plants nine plants (representing fourindependent callus lines) yielded 51 kernels, 31 of which producedvigorous R₁ seedlings (Table 8). Most kernels that developed on SC716plants did not require embryo rescue. Kernels often developed for 30-40days on the plant and some were germinated in soil. The remaining seedwas germinated on MS-based medium to monitor germination and transferredto soil after a few days. In addition to the improved kernel developmentobserved on SC716 R₀ plants relative to SC82 R₀ plants, pollen dehiscedfrom anthers of several SC716 plants and some of this pollen germinatedin vitro (Pfahler, 1967). Transmission of the foreign gene has occurredboth through SC716 R₁ ears and using SC716 R₁ -derived pollen onnon-transformed ears.

                                      TABLE 8                                     __________________________________________________________________________    Regenerated Plants (R.sub.o) and Progeny (R.sub.1)                                     # of                                                                     Independent # of                                                              bar Regenerable # of # # of R.sub.o # of # of                               Exp. Culture Transformants Transformed R.sub.o Reaching Producing                                                    Kernels R.sub.1                        # Bombarded Recovered Callus Lines Plants Maturity Kernels Recovered                                                 Plants                               __________________________________________________________________________    1, 2                                                                             SC82  7      4     76  73   23   153  40                                     4, 5 SC716  20 10  219  (35)  (9) (51)  (31)                                  3 SC94 8 2.sup.a 11.sup.a (0) (0) (0)  (0)                                    6 SC82 19 4.sup.a 23.sup.a (0) (0) (0)  (0)                                 __________________________________________________________________________     .sup.a Regeneration in progress.                                              () Experiment still in progress, data still being collected.             

To date fertile plants from 267 transgenic lines have produced over 59,577 seed (about 227 R₁ seed per transgenic line). Table 2 indicates thatthese plants were derived from 11 different cell lines. In addition bothmale and female fertility has been observed in many of these cellslines. Kernels routinely mature on plants for which the transformant iseither the male or the female parent. Embryo rescue is only necessaryunder unusual circumstances.

Pollen obtained from transformed R₁ plants has been successfullyemployed to pollinate B73 ears and a large number of seeds have beenrecovered (see FIG. 7C). Moreover, a transformed ear from an R₁ plantcrossed with pollen from a non-transformed inbred plant is shown in FIG.7D. The fertility characteristics of the R₁ generation has beenconfirmed both from a standpoint of the pollen's ability to fertilizenon-transformed ears, and the ability of R₁ ears to be fertilized bypollen from non-transformed plants. Fertility of transgenic plants hasbeen maintained for at least 12 generations.

By providing fertile, transgenic offspring, the practice of theinvention allows one to subsequently, through a series of breedingmanipulations, move a selected gene from one corn line into an entirelydifferent corn line without the need for further recombinantmanipulation. Movement of genes between corn lines is a basic tenet ofthe corn breeding industry, involving simply backcrossing the corn linehaving the desired gene (trait). Introduced transgenes are valuable inthat they behave genetically as any other corn gene and can bemanipulated by breeding techniques in a manner identical to any othercorn gene. Exemplary procedures of this nature have been successfullycarried out by the inventors. In these backcrossing studies, the genefor resistance to the herbicide Basta®, bar, has been moved from twotransformants derived from cell line SC716 and one transformant derivedfrom cell line SC82 into 18 elite inbred lines by backcrossing. It ispossible from these 18 inbreds to make a large number of hybrids ofcommercial importance. Eleven of the possible hybrids have been made andare being field tested for yield and other agronomic characteristics andherbicide tolerance. Additional backcrossing to a further 68 eliteinbred lines is underway.

EXAMPLE 33 Analysis of Progeny (R₁) of Transformed R₀ Plants for PAT andBar

A total of 40 progeny of E2/E5 R₀ plants were analyzed for PAT activity,ten of which are shown in FIG. 11A. Of 36 progeny which were assayed, 18had PAT activity. Genomic DNA from the same ten progeny analyzed for PATactivity was analyzed by DNA gel blot hybridization for the presence ofbar as shown in FIG. 11B. The six progeny tested that expressed PATcontained a single copy of bar identical in mobility to that detected incallus and R₀ plants; the four PAT-negative progeny tested did notcontain bar-hybridizing sequences. In one series of assays, the presenceof the bar gene product in 18 of 36 progeny indicates a 1:1 segregationof the single copy of bar found in E2/E5 R₀ plants and is consistentwith inheritance of PAT expression as a single dominant trait. Adominant pattern of inheritance would indicate the presence in the plantof at least one copy of the gene coding for PAT. The single progenyrecovered from an E10 R₀ plant tested positive for PAT activity.

It was determined that the methods disclosed in this invention resultedin transformed R₀ and R₁ plants that produced functionally active PAT.This was determined by applying Basta (PPT) to the leaves of plants anddetermining whether necrosis (tissue destruction) resulted from thisapplication. If functionally active PAT is produced by the plants, theleaf tissue is protected from necrosis. No necrosis was observed on R₀plants expressing high levels of PAT (E2/E5) or on plants expressing lowlevels (E3/E4/E6) (FIG. 8A).

Herbicide was also applied to leaves of R₁ progeny segregating for bar.In these studies, no necrosis was observed on R₁ plants containing andexpressing bar, however, necrosis was observed on those R₁ plantslacking the bar gene. This is shown in FIG. 8B.

Segregation of bar did not correlate with the variability in phenotypiccharacteristics of R₁ plants such as plant height and tassel morphology.In FIG. 5B, the plant on the right contains bar, the plant on the leftdoes not. In addition, most plants were more vigorous than the R₀plants.

Of the 23 R₁ seedlings recovered in this experiment from the SC716transformants, ten of 16 had PAT activity. PAT activity was detected infour of ten progeny from R₀ plants representing callus line R18 and sixof six progeny from R₀ plants representing callus line R9.

O. Embryo Rescue

In cases where embryo rescue was required, developing embryos wereexcised from surface disinfected kernels 10-20 days post-pollination andcultured on medium containing MS salts, 2% sucrose and 5.5 g/l Seakemagarose. Large embryos (>3 mm) were germinated directly on the mediumdescribed above. Smaller embryos were cultured for approximately 1 weekon the above medium containing 10⁻⁵ M abscisic acid and transferred togrowth regulator-free medium for germination. Embryos that becamebacterially contaminated; these embryos were transferred to mediumcontaining 300 μg/ml cefoxitin. Developing plants were subsequentlyhandled as described for regeneration of R₀ plants.

EXAMPLE 34 Embryo Rescue

Viable progeny, recovered from seven SC82 E2/E5 plants and one SC82 E10plant, were sustained by embryo rescue. This method consisted ofexcising embryos from kernels that developed on R₀ plants. Embryosranged in size from about 0.5 to 4 mm in length. Small embryos werecultured on maturation medium containing abscisic acid while largerembryos were cultured directly on germination medium. Two of theapproximately forty viable progeny recovered from SC82 R₀ plants byembryo rescue are shown in FIG. 7B.

P. Phenotype of Transgenic Plants

Most of the R₀ plants regenerated from SC82 transformants exhibited anA188×B73 hybrid phenotype. Plants were similar in height to seed derivedA188 plants (3-5 feet) but had B73 traits such as anthocyaninaccumulation in stalks and prop roots, and the presence of uprightleaves. Many plants, regardless of the callus line from which they wereregenerated, exhibited phenotypic abnormalities including leafsplitting, forked leaves, multiple ears per node, and coarse silks.Although many of the phenotypic characteristics were common to all R₀plants, some characteristics were unique to plants regenerated fromspecific callus lines. Such characteristics were exhibited regardless ofregeneration route and the time spent in culture during regeneration.

Nontransformed control plants were not regenerated from this cultureand, therefore, cannot be compared phenotypically. Pistillate flowersdeveloped on tassels of one E11 (1/6 ), several E10 (3/22 ) and almostone-third of the E2/E5 (12/37 ) plants with a range of three toapproximately twenty ovules per tassel. Primary and secondary earsdeveloped frequently on most E2/E5, E10, and E11 plants; a mature E2/E5plant is shown in FIG. 7A. Anthers rarely extruded from the tassels ofplants regenerated from SC82 transformants and the limited number ofanthers which were extruded did not dehisce pollen. Some phenotypiccharacteristics observed were unique to plants regenerated from aspecific callus line such as the lack of ears on E3/E4/E6 plants and a"grassy" phenotype (up to 21 long narrow leaves) exhibited by all E11plants.

All SC82 plants senesced prematurely; leaf necrosis began approximatelytwo weeks after anthesis. The R₀ plants regenerated from SC82transformed cell lines have tended to senesce prematurely; typicallybefore the developing kernels were mature. This has necessitated the useof embryo rescue to recover progeny (R₁ generation). Segregation of barin the R₁ generation does not correlate with the variability inphenotypic characteristics of R₁ plants such as plant height and tasselmorphology. In FIG. 7B, the plant on the right contains bar, the planton the left does not. In addition, most of the R₁ plants are morevigorous than the R₀ plants. Transformed progeny (R₁) have producedkernels and progeny testing has now been advanced to the R₁₂ generation.

Of 219 plants regenerated from 10 independent SC716 transformants,approximately 35 reached maturity (Table 8). The SC716 plants did notexhibit the phenotypic differences which characterized the plantsregenerated from the individual callus lines of SC82. These plants weremore uniform and abnormalities less frequent. The phenotype of theseplants closely resembled that of control plants regenerated from a SC716cryopreserved culture which was not bombarded. Plant height ranged fromthree to six feet with the majority of the plants between five and sixfeet. Most mature plants produced large, multi-branched tassels andprimary and secondary ears. Pistillate flowers also developed on tasselsof several SC716 plants. Although anther extrusion occurred atapproximately the same low frequency as in the SC82 plants, a smallamount of pollen dehisced from some extruded anthers. For most of theSC716 plants that reached maturity, senescence did not commence until atleast 30 days after anthesis.

The improved characteristics of SC716 plants over SC82 plants indicatethat differences between the suspension cultures may be responsible.This observation has been supported by further experiments in whichAT824 plants have been regenerated. These plants are normal inappearance. Plants produce normal tassels and shed viable pollen. Inaddition plants do not prematurely senesce and seed will mature on theplant. Many plants derived from this cell line and the lines ABT4 andHi-II are indistinguishable from nontransformed plants.

I. Transformation with Genes for Desirable Traits

One of the distinct advantages provided by the present invention is theability to transform monocot plants, such as maize, with a gene or geneswhich imparts a desirable trait to the resultant transgenic plants.These traits include, for example, resistance to insects, herbicides,drought, etc., and the improvement of characteristics such asappearance, yield, nutritional quality, and the like. Certain such geneswhich are highly desirable in monocot transformation have been discussedas selectable markers, for example, bar and EPSPS. These genes encodeproteins which confer herbicide resistance on the plant. Otherparticularly preferred transgenes include those that have insecticidalactivities, such as toxins, proteinase inhibitors and lectins, and thosegenes that alter the nutritional quality of the grain. The followingexamples illustrate the use of the present invention in generatingadvantageous transgenic plants. Table 9 lists all of the genessuccessfully introduced into maize by the inventors and summarizes thestatus of analysis for the presence of the introduced DNA andexpression. The Table indicates that stable transformants have beenrecovered containing all genes attempted. Expression has been detectedfrom all structural genes listed in at least one transformed cell linein studies that have progressed to this stage. Detection of expressionis dependent on the promoter and enhancers used to drive expression ofthe structural gene, the structural gene itself, and the limits of thedetection system. At this point in time fertile plants containing theuidA, bar, Bt, aroA, dapA, 10 kD zein storage protein and hygromycinresistance genes have been recovered from transformants. Expression ofthe bar gene has been detected in progeny from all transformantsexamined (19/19). Expression of the uidA gene has been detected in theprogeny of one out of five transformants assayed.

The protocols employed for preparing the transgenic plants described inthe foregoing Table were as described above. The preparation of thevarious vectors, etc., was accomplished through the application ofmolecular biology techniques as described aboveand/or using routinelaboratory procedures. The numeral designations under "Protocol"represent the following:

1. Tissue (suspension) was plated on filters, bombarded and then filterswere transferred to culture medium. After 2-7 days, the filters weretransferred to selective medium. Approximately 3 weeks afterbombardment, tissue was picked from filters as separate callus clumpsonto fresh selective medium.

2. As in 1above, except after bombardment the suspension was put backinto liquid--subjected to liquid selection for 7-14 days and thenpipetted at a low density onto fresh selection plates.

3. Callus was bombarded while sitting directly on medium or on filters.Cells were transferred to selective medium 1-14 days after particlebombardment. Tissue was transferred on filters 1-3 times at 2 weeksintervals to fresh selective medium. Callus was then briefly put intoliquid to disperse the tissue onto selective plates at a low density.

4. Callus bombardment. The tissue was transferred onto selective platesone to seven days after DNA introduction. Tissue was subcultured assmall units of callus on selective plates until transformants wereidentified.

                                      TABLE 9                                     __________________________________________________________________________                                        Gene in                                                                             Progeny                               Expression  Gene in Callus Gene in Ro Ro Plant Progeny Plant                  Cassette Protocol Callus Expression Plant Expression Plant Expression       __________________________________________________________________________    uidA (GUS,                                                                             1, 2, 3, 4                                                                         +   +     +     +     +     +                                     reporter (gene                                                                bar (bialaphos 1, 2, 3, 4 + + + + + +                                         resistance,                                                                   selectable                                                                    marker)                                                                       lux (luciferase 2 + + + + In progress                                         reporter, gene)                                                               hyg 4 + + + + + +                                                             (hygromycin                                                                   resistance,                                                                   selectable                                                                    marker)                                                                       35S-adh-aroA 2 + + + + + +                                                    (Gyphosate                                                                    tolerance)                                                                    α-tubulin-aroA 2 + + + + In                                             (Glyphosate      progress                                                     tolerance)                                                                    2xhis-aroA 2 + - ND ND In                                                     (Glyphosate      progress                                                     tolerance)                                                                    35Shis-aroA 2 + - ND ND In                                                    (Glyphosate      progress                                                     tolerance)                                                                    R, C1 1 + +                                                                   (anthocyarin                                                                  pigment                                                                       synthesis)                                                                    35S-IaB6 (Bt) 2 + ND + ND In                                                        progress                                                                35S-HD73 4 + - + - + ND                                                       (Bt)                                                                          35S-1800b 2, 4 + - + + + +                                                    (Bt)                                                                          2730CS-AdhVI- 2 + ND + + In progress                                          1800b (Bt)                                                                    35S-Adh1-1800b 2, 4 + ND + In                                                 (Bt)     progress                                                             35S-MZTP-1800b 2, 4 + In                                                      (Bt)   progress                                                               Adh1-adh1-1800b 4 + + + - Completed                                           (Bt)                                                                          potato pinII 2 + + + ND In                                                    (proteinase      progress                                                     inhibitor                                                                     confers insect                                                                resistance)                                                                   tomato pinII 2 + ND + ND In                                                   (proteinase      progress                                                     inhibitor                                                                     confers insect                                                                resistance)                                                                   35S-dapA 3, 4 + + In                                                          (altered lysine    progress                                                   production)                                                                   Z27-dapA 3, 4 + NA ND ND In                                                   (altered lysine      progress                                                 production in                                                                 seed)                                                                         Z27Z10 (altered 3, 4 + NA ND NA + +                                           sotrage protein                                                               in seed)                                                                      Z4Z10 (altered 3, 4 + NA ND NA In                                             storage protein      progress                                                 in seed)                                                                      Z10Z10 (altered 3, 4 + NA ND NA In                                            storage protein      progress                                                 in seed)                                                                      10Z4ENT (altered 3, 4 + NA ND NA In                                           storage protein      progress                                                 in seed)                                                                      1020P (altered 3, 4 + NA ND NA In                                             storage protein      progress                                                 in seed)                                                                      35S-adh1-mt1D 2 + In                                                          (enhanced stress   progress                                                   resistance)                                                                   deh (resistance 4 ND + In                                                     to dalapon    Progress                                                        herbicide)                                                                  __________________________________________________________________________     NA indicates not applicable, e.g., gene does not express in that tissue       type.                                                                         ND indicates not done, but tissue was available.                              Blank space indicates experiment has not progressed to this point or was      terminated before this point.                                                 The symbol "+" indicates that expression of the gene was detected by RNA,     protein, enzyme assay or biological assay.                               

1. Herbicide Resistance

EXAMPLE 35 Glyphosate resistance--Tranformants Containing the Salmonellatyphimurium aroA Gene

This example describes certain methods relating to the use of an aroAgene construct in maize transformation. The herbicide glyphosate acts byinhibiting the enzyme EPSP Synthase. EPSP Synthase is presents in plantsand bacteria and the gene used in this example, aroA, was isolated fromSalmonella typhimurium. Certain mutated versions of the aroA gene areknown which encode variant EPSP Synthase enzymes which are insensitiveto glyphosate (Comai et al., 1983).

Transformation studies were conducted employing pDPG238, a tandembar-aroA construct containing a Calgene aroA plant expression cassette.Transformation using the SC716 culture yielded four clones that producedforty-five plants in the greenhouse. Results from PCR analysisdemonstrated that three of these four clones contained the aroA gene andone line also expressed the gene at the limit of detection by Westernanalysis. A plant from this clone was pollinated, two embryos rescuedand two R₁ plants grown (designated TGB-4 and TGB-5). Both R₁ plantscontained the aroA gene as determined by PCR analysis and one plant(TGB-5) expressed the gene as determined by Western analysis. Progeny ofthe aroA expressing plant (TGB-5) were included in field tests.

An experiment was conducted to examine the level of resistance toglyphosate (Roundup®) in crosses of the aroA expressing line TGB-5produced using pDPG238. This line contains both the bar and aroA genesand hence is expected to confer resistance to both Basta® and Roundup®.A single progeny of TGB-5, designated TGB-56, was crossed to four eliteinbreds, representing four different heterotic groups. The progeny weregrown and self-pollinated to increase seed, and the resultant seeds wereplanted in two experiments. The first experiment was sprayed with 1.5lb/A Basta® to confirm Mendlian segregation of the introduced DNA (Table10). The remaining plants were divided into four blocks and sprayed withapplication rates of Roundup® of 2 oz/A, 4 oz/A, 8 oz/A, and 16 oz/A.All plants were killed in the 8 oz and 16 oz treatments (normal fieldapplication rates for weed control). At 4 oz/A a portion of thetransformed plants were killed and a portion were stunted in growth. Theratio of dead plants to slow growing plants was not significantlydifferent from the expected 3:1 ratio in progeny from three of the fourself pollinations (Table 11), indicating that there was a low level ofexpression of the aroA gene providing partial resistance to herbicideapplication.

TABLE 10: Segregation of Basta® resistance.

Resistant Susceptible

(CD×TGB-56)12856χ² =1.22

(AW×TGB-56)12952χ² =1.10

(CN×TGB-56)13648χ² =0.075

(AF×TGB-56)10744χ² =1.17

                  TABLE 11                                                        ______________________________________                                        Segregation of Roundup ® Resistance.                                                  Resistant   Susceptible                                           ______________________________________                                        (CD × TGB-56)                                                                       63          26        χ.sup.2 = 0.739                           (AW × TGB-56) 66 30 χ.sup.2 = 1.680                                 (CN × TGB-56) 64 25 χ.sup.2 = 0.005                                 (AF × TGB-56) 53 30 χ.sup.2 = 4.430                               ______________________________________                                    

Studies involving the E1 cell line yielded nine clones that haveproduced sixty-five plants in the greenhouse. Results from PCR analysisdemonstrated that five of these clones contain aroA, and Westernanalysis of leaf tissue indicated that certain plants (clone #58)express aroA. No R₁ progeny were recovered from E1 plants.

Several tandem bar-aroA vectors were utilized in which aroA expressionunits had been introduced into pDPG295 and pDPG298, as described earlier(see section E, DNA segments). Briefly, these include vectors with a35S-histone promoter fusion, in which the genes are placed inconvergent, divergent, and colinear orientations (pDPG314, pDPG313, andpDPG317, respectively) with respect to the bar expression cassette;colinear and divergent vectors employing a histone promoter (pDPG315 andpDPG316, respectively); and colinear and divergent vectors employing anα-tubulin promoter (pDPG318 and pDPG319, respectively). DNA of five ofthese constructs was prepared for bombardment (pDPG313, pDPG314,pDPG315, pDPG317, pDPG319).

It was firstly determined which of the tandem bar-aroAorientation-promoter combinations functioned best under the experimentalemployed. The 5 DNA constructs, pDPG313, pDPG314, pDPG315, pDPG317, andpDPG319, were bombard into the E1 cell line. Many transformed cloneswere recovered which revealed that no construct was more useful than anyother using bialaphos selection. PCR analysis showed thatco-transformation frequencies were about identical (75%) regardless ofwhich construct was used. It was therefore concluded that all constructswere functioning, and that no one construct was better than another fortransformation. Clones from these studies were cryopreserved for futureanalysis.

Since all constructs appeared to be functioning similarly, efforts wereconcentrated on using the 3 divergently constructed tandems pDPG315,pDPG317, and pDPG319 in which the aroA gene is driven by the 2× histone,Camv 35S-histone, and α-tubulin promoters, respectively. Bombardmentswere initiated using 16 new cell lines (other than E1). These linesinclude AT824, SC716, E4, ABT4, and various other new A188×B73 andB73×A188 cultures. aroA transformants were successfully recovered fromcell lines AT824, SC716, and E4 (A1880 cell culture revived from EniMontcryopreservation). Regeneration was begun on aroA confirmed clones ofall three lines. Regenerated plants from the AT824 and SC716 clones arein the greenhouse. Four SC716 transformants containing the α-tubulinpromoter-aroA expression vector produced R₁ seed. AT824 transformantsproduced R₁ seed as follows: five transformants containing the α-tubulinpromoter, two transformants containing the CaMV 35S-histone fusionpromoter and six transformants containing the 2× histone promoter. Thesetransformants are currently in field tests to determine levels ofglyphosate resistance.

EXAMPLE 36 Tissue Specific Expression of aroA in Roots of TransgenicPlants

Transformants were maintained on medium 223 (Table 1). For regenerationcells were transferred to medium 189 (Table 1) and cultured in the dark.Cultures were subcultured two weeks later onto fresh medium 189 (Table1). Regenerating tissue was transferred to medium 101(Table 1) in lowlight, followed by rooting of shoots on medium 607 (Table 1) or 501(Table 1) and transfer to Plant Cons®. Rooted plants were grownhydroponically to avoid soil and microbial contamination when attemptingto assay plants for root specific expression of the EPSPS gene.

Expression of the EPSPS gene, aroA, was assayed by Western blotanalysis. Leaf and root samples were harvested from transgenic plants.Approximately 1 gram of tissue was ground in a glass homogenizer with400 ul RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris-HCl pH8.0). Extracts were centrifuged at 14,000×g and supernatants collectedfor protein analysis. Forty ul of each protein extract was run on a12.5% polyacrylamide denaturing gel (Laemmli, 1970). The gel was runovernight at 50 volts. Following running the gel was electroblotted tonitrocellulose paper and the nitrocellulose dried at 37° C. The blot waswashed in 50 ml 5% nonfat dry milk (NFD) in TBS (20 mM Tris-HCl pH 7.5,0.5M NaCl) for 30-60 minutes followed by incubation overnight in 50 ml5% NFD, TBS containing 100 ul EPSPS rabbit antiserum. The nitrocelluloseblot was washed 3 times for 5 minutes each in TBS and then incubated in50 ml 5% NFD, TBS containing 100 ul goat anti-rabbit antiserumconjugated to horseradish peroxidase for two hours. The nitrocelluloseblot was washed 3 times with TBS for 5 minutes each prior to staining.The blot was stained as follows. Twenty four mg 4-chloro-1-naphthol wasdissolved in 8 ml methanol. Forty two ul 3% H₂ O₂ was added. Thirtyminutes after initiation of staining 400 ul of H₂ O₂ was added. Thestaining reaction was stopped by adding water and drying the blot.Western blots were stored in the dark.

Expression of EPSPS was detected in roots derived from plants oftransformant S10AV13. No expression was observed in leaf tissue. Theprotein expressed in the root was identical in size to EPSPS proteinisolated from Salmonella tyhphimurium and run on the same gel as apositive control. This is the expected expression profile expected,because the α-tubulin promoter is root specific in maize. The aroAexpression cassette in pDPG319 also contains a transit peptide of about130 amino acids to target the EPSPS protein to the plastids. As theprotein expressed in maize is identical in molecular weight to theprotein isolated from S. typhimurium it is apparent that the transitpeptide has been correctly cleaved from the EPSPS protein. Hence it isbelieved that the EPSPS protein was targeted to the plastids asdemonstrated in example 7.

EXAMPLE 37 Herbicide Application (Basta®)

The herbicide formulation used, Basta TX®, contains 200 g/l glufosinate,the ammonium salt of phosphinothricin. Young leaves were painted with a2% Basta solution (v/v) containing 0.1% (v/v) Tween-20. The prescribedapplication rate for this formulation is 0.5-1%.

In FIG. 8A, Basta® solution was applied to a large area (about 4×8 cm)in the center of leaves of a nontransformed A188×B73 plant (left) and atransgenic R₀ E3/E4/E6 plant (right). In FIG. 8B, Basta was also appliedto leaves of four R₁ plants; two plants without bar and two plantscontaining bar. The herbicide was applied to R₁ plants in 1 cm circlesto four locations on each leaf, two on each side of the midrib.Photographs were taken six days after application.

EXAMPLE 38 Resistance in the Field to the Herbicide Ignite®/Basta®

Experiments have been undertaken to determine whether transformantscontaining the bar gene exhibit sufficient levels of herbicideresistance to be useful commercially. Eleven independent bartransformants were evaluated for field levels of resistantce to theherbicide Basta® (a.k.a. Ignite®). The field design used a split-splitplot with 2 repetitions. Whole plots were spray rates (1×, 3× and 7×)and subplots were transformant sources. Transformant sources wereplanted in four-row plots with two rows sprayed with Ignite and two rowssprayed with water. A spray rate of 0.331b/A (1×) was used to test forefficacy. This will probably be the field rate for weed control in cornfields.

The range of responses was too narrow and field variation too large fora rating system to be useful for evaluating field levels of resistance.At the normal field application rate of Ignite® all transformant sourcesdemonstrated resistance to the herbicide. Differences were observedbetween the transformants at an application rate that was three timesthe normal rate. Each plot was examined by comparing sprayed versusunsprayed rows for 1) leaf-necrosis in the whorl where the herbicideaccumulated at spraying; 2) chlorosis of the whorl tissue; 3) abnormalleaf growth in the whorl after spraying; 4) variability and stunting ofsprayed planting compared to unsprayed; and 5) overall reduction inplant growth of sprayed versus unsprayed rows.

Differences in herbicide sensitivity were not dramatic, but consistentenough to rank each transformant , i.e. all transformant showedresistance to the herbicide. Transformants could be roughly classifiedinto three major response groups with little difference between sourceswithin the group . Transformants A24, B16 and E29 were most resistant tothe herbicide. The sources A24 and B16 were the best, with no phenotypicdifference between sprayed and unsprayed plots. Source E29 was also verygood, but was somewhat variable or slightly shorter in sprayed plots.Sources A18, V11, and E19 were intermediate in response to Ignite®. Thesprayed rows were slightly shorter, but were uniform and did not haveany of the phenotypic abnormalities seen in the more sensitivetransformants. Transformant sources G18, G20, K20, E14 and E27 wereclearly sensitive to the 3× application rate of Ignite®. They wereshorter and had plants with necrotic lesions, chlorotic whorls and leafabnormalities group.

These experiments cleary demonstrate that transformants containing thebar gene are useful for production of herbicide resistant commercialhybrids.

2. Grain Quality

EXAMPLE 39 Elevation of Lysine Levels in Maize Grain

As described in the previous U.S. patent application Ser. No. 07/204,388(most claims of which have been granted), one approach to enhancinglysine levels in maize grain involves lysine overproduction throughderegulation of the lysine biosynthetic pathway. A key regulatory pointin the lysine biosynthetic pathway occurs at the condensation reactionin which pyruvate and aspartyl semialdehyde form dihydrodipicolinicacid. This reaction is catalyzed by the enzyme dihydrodipicolinic acidsynthase (DHDPS), which is normally feedback-inhibited by free lysine.In that previous patent application, data were presented whichdemonstrate that expression of a lysine-tolerant version of DHDPS,encoded by the E. coli dapA gene, in transgenic tobacco plants leads toelevated lysine levels in plant cells. As presented below, we havetransferred similar gene constructs to maize cells and have successfullyregenerated transgenic plants which contain these dapA gene constructsand express the lysine-tolerant DHDPS in maize seeds.

Plasmid constructs were introduced, in various combinations, into maizecells by particle bombardment as described above. Transgenic cell lineswere identified on the basis of resistance to the appropriate selectableagent, either hygromicin (Hyg), phosphoinothricin (Ppt), or bialophos(Blp), included in the growth medium. These lines were then screened atthe callus level for presence of appropriate DNA sequences by PCRamplification assays. Several cell lines have been established which areat various stages of plant regeneration. Current status of transformedplants that have been transferred to soil is summarized in the followingTable 12:

                  TABLE 12                                                        ______________________________________                                                                     Transgenic                                         DPG   line(s)                                                                 plasmid   carried                                                             constructs Recipient  through                                                 used for cell Selection plant                                                 bombardment line agent regeneration Status                                  ______________________________________                                        334/367   AB61     Hyg       HAL       R2                                           seed                                                                      334/366 AB61 Hyg Dap2 R2                                                          plants                                                                    334/371/367 AB61 Hyg Dap3 R2                                                      plants                                                                    335/355 HAL Ppt Dap4 R1                                                           seed                                                                      371/363 ABT4 Ppt ND1-1 R1                                                        ND1-4 plants                                                               335/372/231 ABT4 Blp dAH03CF-10 R0                                               dAH06CF-10 plants                                                             dAH06CF-15                                                                    dAH09CF-11                                                                 335/372/231 ABT4 Blp dAH04CG-11 R0                                            335/372/283   dAH07CF-11 plants                                                  dAH07CF-12                                                                    dAH07CF-15                                                                    dAH07CF-19                                                                    dAH07CF-24                                                                    dAH07CG-17                                                                 335/165 HB13-3 Blp dAU01CG-10 R0                                                  plants                                                                  ______________________________________                                    

For each of the most recent experiments, where only R0 plants have beengenerated to date, several additional cell lines are in the plantregeneration process. In these experiments, in which the main objectiveis to obtain expression of the DSTP/dapA transgene in various tissues,several dozen transformants have been obtained by co-bombardment withmultiple plasmids. Only those lines which, when assayed at the calluslevel, are PCR-positive for the selectable marker transgene and the dapAconstruct(s) of interest are transferred to the plant regenerationprogram. In summary, these experiments have yielded the followingnumbers of bar-positive transformants:

                  TABLE 13                                                        ______________________________________                                        Genotype by PCR assay                                                                          No. transformants                                            ______________________________________                                        DPG 335/372      35                                                             DPG 335 21                                                                    DPG 372 26                                                                    DPG 418 20                                                                  ______________________________________                                    

PCR-amplification assays of DNA extracted from callus samples wereperformed by standard procedures. In each assay, one oligonucleotideprimer (primer 1 in the table below) was specific to the promoterpresent in the transgene, and a second primer (primer 2) corresponded tothe dapA coding region or, for pDPG418, the Glb1 3' sequence.Designations of the primers used for the PCR-amplification assays of thedapA constructs, along with the sizes of the amplified fragmentproducts, are provided in the following Table 13:

                  TABLE 13                                                        ______________________________________                                        Construct    Primer 1  Primer 2  Fragment size                                ______________________________________                                        334          Z10P965   dapAPCR1   572 bp                                        Z10/MZTP/dapA/nos                                                             335 Z27mid DAP6 1011 bp                                                       Z27/DSTP/dapA/35S                                                             371 35SPCR1 dapAPCR1  480 bp                                                  35S/MZTP/dapA/nos 35SPCR1 DAP6  610 bp                                        372 35SPCR1 dapAPCR1  486 bp                                                  35S/DSTP/dapA/nos 35SPCR1 DAP6  616 bp                                        418 Glb15' Glb13' 1300 bp                                                     Glb1/DSTP/dapA/G1 Glb15' dapAPCR1  464 bp                                     b1 Glb15' DAP6  594 bp                                                      ______________________________________                                    

Expression of dapA transgenes in transformed plant cells has beenanalyzed to date primarily by assaying for the presence oflysine-tolerant DHDPS activity essentially as described in U.S. patentapplication Ser. No. 07/204,388, except that modifications have beenmade for use of the assay in a qualitative manner in a microtiter plateformat as follows: a few milligrams of each tissue sample are placed inthe wells of a 96-well plate with 50 ul of 0.225 M tris, 17.3 mM sodiumpyruvate, pH8.2, covered, then transferred to a -20° C. freezer for atleast one hour. After the samples are thawed briefly, 50 ul of thereaction mix described by Yugari et al (J. Biol. Chem, 240:4710-4716,1965), supplemented with L-lysine to 0.45 mM, was added and thereactions were incubated at 37° C. for 1-2 hours. Reactions werequenched by the addition of 175 ul stop buffer, and pink color wasallowed to develop at room temperature for 15-60 minutes, at which timethe samples were scored as plus or minus lysine-tolerant DHDPS activityon the basis of presence or absence, respectively, of pink color. Thisassay has been applied in this form to portions of cultured maizecallus, portions of immature seeds, fragments of mature dry kernels, andleaf sections from plants at various stages of growth. Results oflysine-tolerant DHDPS expression assays in the transgenic maize linesfrom which mature plants have been obtained are summarized in thefollowing Table 14:

                  TABLE 14                                                        ______________________________________                                                 dapA                    Immature                                       Transgenic promoter   kernels Mature                                          line construct(s) Callus Leaf (20 DAP) kernels                              ______________________________________                                        HAL      334 (Z10) no      nd    nd     nd                                      Dap2 334 (Z10) no no yes yes                                                  Dap3 334 (Z10) nd no yes yes                                                   371 (35S)                                                                    Dap4 335 (Z27) nd nd nd nd                                                    ND1-1 371 (35S) yes nd nd n.d.                                                ND1-4 371 (35S) yes yes nd no                                               ______________________________________                                    

The expression of the dapA transgene in both developing and mature maizekernels from transgenic plants is significant with respect to use ofthese, and related, gene constructs in development of high-lysine maizetypes through deregulation of the lysine biosynthetic pathway. Thistrait is transmitted and expressed at least through the R2 generation,as R2 seeds of both Dap2 and Dap3 contain lysine-tolerant DHDPSactivity.

The following additional lines have been assayed for lysine-tolerantDHDPS activity at the callus level. These lines have either producedplants that have recently been transferred to soil or are in the earlystages of plant regeneration (Table 15).

                  TABLE 15                                                        ______________________________________                                                     dapA      Lys-tolerant                                             Cell line construct(s) DHDPS activity                                       ______________________________________                                        dAH03CF-10   335/372   +                                                        dAH06CF-10  +                                                                 dAH06CF-15  -                                                                 dAH09CF-11  -                                                                 dAH04CG-11 335/372 -                                                          dAH07CF-11  -                                                                 dAH07CF-12  +                                                                 dAH07CF-15  -                                                                 dAH07CF-19  -                                                                 dAH07CF-24  -                                                                 dAH07CG-17  +                                                                 dAU01CG-10 335 +                                                              dAR01E1-10 418 -                                                              dAR01E1-11  +                                                                 dAR01E1-13  +                                                                 dAR01E1-14  +                                                                 dAR01E1-15  -                                                                 dAR01E1-16  +                                                                 dAR01E1-17  -                                                                 dAR01E1-18  +                                                                 dAR01E1-19  +                                                                 dAR01E1-20  +                                                                 dAR01E1-21  +                                                                 dAR01E1-23  -                                                                 dAR01E1-24  -                                                               ______________________________________                                    

Expression of the dapA transgene in seeds of transformed plants asdescribed above is very encouraging with respect to our goal ofderegulating lysine biosynthesis in maize kernels. To date, expressionof the lysine-tolerant dapA gene product has been accomplished by usingthe endosperm-specific promoters Z10 and Z27, and it is anticipated thatuse of the embryo-specific Glb1 promoter will result in expression ofthe dapA gene product in embryos as well.

EXAMPLE 40 Enhanced Methionine Content of Maize Seeds

The purpose of these experiments is to enhance methionine content ofmaize kernels for improved poultry feed. This goal is achieved throughparticle bombardment of maize cells with DNA-coated microprojectiles andsubsequent selection of transformed cells, followed by regeneration ofstably transformed, fertile transgenic maize plants which transmit theintroduced genes to progeny. The gene used for enhanced methioninecontent encodes a 10 kD zein seed storage protein which is 23%methionine, and seeds of transformed plants overexpress this gene,leading to increased 10 kD zein and increased methionine content.

The zeins are a large family of related proteins which accounts for morethan 50% of the total protein in maize seeds. The α-zeins, which are lowin lysine, methionine and tryptophan, are the most abundant of thezeins. Thus, maize seeds are deficient in these amino acids because sucha large fraction of the total protein is α-zein. One method to correctfor this deficiency, and to substantially increase the seed levels ofvarious amino acids, especially methionine, is to overexpress a geneencoding a 10 kD δ-zein containing 23% methionine.

U.S. patent application Ser. No. 07/636,089, filed Dec. 28, 1990,describes the production of transgenic Zea mays plants and seeds, whichhave been transformed with recombinant DNA encoding the 10 kD δ-zein.Transgenic plants are obtained by bombardment of friable, embryogeniccallus with microprojectiles coated with recombinant DNA encoding the 10kD zein and a selectable marker gene, followed by selection oftransformed callus and regeneration of fertile plants, which transmitthe introduced gene to progeny.

A transformed cell line, designated Met1, was obtained by bombardingAB63S cells with the plasmids pDPG367, pDPG338, and pBII221. Selectionwas on 60 mg/l hygromycin, and the presence of the HPT, GUS and Z27Z10genes was confirmed by PCR analysis. Additionally, the presence of theHPT coding sequence and the Z27Z10 gene was confirmed by Southernanalysis. Met1 exhibited strong resistance to hygromycin, and was only20% inhibited at 200 mg/l hygromycin. Thirty two additional linescarrying methionine constructs were identified (using selectionprocedures described elsewhere in this CIP) as shown in Table 16.

                  TABLE 16                                                        ______________________________________                                        Genotypes of Cell Lines PCR.sup.+  for Methionine                               Constructs                                                                         Cell Line     Genotype                                                 ______________________________________                                        Met1             Z27Z10                                                         MD 64-1 Z27Z10, Z10Z10                                                        MD 84-34 Z27Z10, Z4Z10                                                        MD 84-31 Z4Z10, Z10Z10                                                        MD 84-2 Z4Z10                                                                 MD 52-8 Z4Z10, Z10Z10                                                         MD 52-11 Z27Z10, Z4Z10, Z10Z10                                                MD 52-10 Z4Z10                                                                MD 44-2 Z4Z10, Z10Z10                                                         MD 42-1 Z27Z10, Z4Z10, Z10Z10                                                 MD 32-2 Z10Z10                                                                MD 32-1 Z27Z10, Z10Z10                                                        A6-101 Z27Z10, Z4Z10, Z10Z10                                                  A6-115 Z27Z10, Z4Z10, Z10Z10                                                  A6-181 Z27Z10, Z10Z10                                                         A6-151 Z27Z10, Z4Z10, Z10Z10                                                  A6-161 Z4Z10, Z10Z10                                                          A6-907 Z27Z10, Z4Z10, Z10Z10                                                  A8-113 Z10Z10                                                                 A10-2 Z27Z10, Z4Z10, Z10Z10                                                   A10-7 Z27Z10, Z4Z10, Z10Z10                                                   B1-72 Z27Z10                                                                  B1-82 Z27Z10                                                                  B1-94 Z27Z10                                                                  B1-101 Z27Z10                                                                 B1-401 Z27Z10                                                                 B1-691 Z27Z10                                                                 B1-702 Z27Z10                                                                 B1-703 Z27Z10                                                                 B1-703 Z27Z10                                                                 B1-704 Z27Z10                                                                 B1-705 Z27Z10                                                                 B1-901 Z27Z10                                                               ______________________________________                                    

Transformants were identified by PCR analysis. The Z27Z10 chimeric genewas distinguished from endogenous genes by generation of a-PCR productwhich spanned the junction of the Z27 promoter and the Z10 codingsequence. Similarly, the Z10Z10 introduced gene was identified by a PCRproduct which spanned the junction of the pUC plasmid which carried theconstruct and the Z10 promoter, and the Z4Z10 construct was identifiedby a PCR product spanning the junction between the Z4 promoter and theZ10 coding sequence.

Forty five plants were regenerated from Met1 callus. These plants wereselfed and reciprocal crosses were made using 6 inbred lines. Immatureseed was harvested at 21-24 DAP for Northern analysis, and genotype ofthe progeny was also examined by PCR. In all cases, the HPT and Z27Z10genes cosegregated, consistent with Mendelian segregation of a singlelocus, and indicating linkage of the introduced HPT and chimeric Z27Z10genes. Additionally, the GUS gene was shown to cosegregate with the HPTand Z27Z10 genes, allowing the use of a GUS assay to be used to identifythe Z27Z10 genotype. Northern analysis confirmed that the chimericZ27Z10 gene was expressed only in seeds PCR⁺ for the Z27Z10 gene. Aswith PCR analysis, the presence of the Z27Z10 transcript was confirmedusing a probe which spanned the junction between Z27 and Z10 sequences.

ELISA analysis of Met1 R₁ seed using a Z10-specific antibody revealed atrend of increased 10 kD zein levels as compared to controls. Seedscarrying the Z27Z10 gene showed 2 to 3-fold higher levels of 10 kD zeinper unit protein than nontransformed seed. Even more striking resultswere obtained in ELISA analysis of R₂ seeds. In these experiments,embryos were isolated from seeds and germinated, and PCR analysis forthe Z27Z10 gene was carried out on the seedlings from the excisedembryos, and protein analysis was carried out on the remaining seedtissue. The Z10 gene product accounted for up to 0.6% of the dry seedweight, or 6% of the total protein. An average 7-fold increase in 10 kDzein expression was found. Field test data from 90 bulked R₃ seedsamples indicated a positive correlation between elevated 10 kD zeinlevels and the presence of the Z27Z10 construct. For ELISA analysis,zeins were extracted from corn meal samples in 70% ethanol and 2%B-mercaptoethanol at room temperature. Extracts were dried down on96-well microtiter plates and incubated sequentially with 1% BSA,primary antibody, peroxidase-conjugated secondary antibody, and enzymesubstrates (for peroxidase). Absorbances at 490 minus 410 nanometerswere collected, and a standard curve using purified 10 kd-specificantibody was used to calibrate each plate. Three extractions werecarried out for each sample, and each extraction was assayed in 3 wells.Thus, 9 absorbance measurements were made for each sample.

Protein levels were measured by near infra-red reflectance spectroscopy,and methionine levels were measured by oxidation of meal, followed byacid hydrolysis with detection of the released methionine sulfone byPITC (phenyl-isothiocarbymate) pre-column derivitization andreverse-phase HPLC. The presence of Z27Z10 DNA was confirmed by PCRand/or Southern analysis.

PCR, 10 Kd zein, methionine and protein analyses were carried out onfield test samples from 65 ears. Of the three lines analyzed, it wasdemonstrated that PCR⁺ ears contained higher levels of 10 Kd zein,regardless of genotype. In addition, a correlation was shown betweenincreased Z10 gene product, as determined by ELISA analysis, andelevated methionine levels in the seed, as determined by amino acidanalysis.

EXAMPLE 41 Improved Protein and Starch Content of Maize Seeds byAntisense DNA

The purpose of these experiments is to improve the nutritional contentof maize kernels by reducing the expression of α-zeins, with aconcommitant increase in the levels of other proteins or starch. Thereduction in α-zein expression is to be achieved by particle bombardmentof maize cells with microproprojectiles coated with antisense genes tothe 19 and 22 kD α-zein families, to reduce translation of α-zein mRNA.

The majority of the zein proteins, which account for over 50% of thetotal seed protein, are the 19 and 22 kD α-zeins. The high levels ofα-zeins, which are low in methionine, lysine and tryptophan, result inseeds low in these amino acids. Maize seed protein levels are inverselycorrelated with starch, thus reduced α-zein levels would potentiallyresult in increased starch. Increased levels of other proteins are alsoassociated with reduced levels of α-zeins in opaque and floury mutants,which reduce levels of α-zeins.

The α-zeins are encoded by a large multigene family with regions ofsequence homology. Consequently, a small number of introduced antisensegenes (with transcripts complementary to the conserved regions ofhomology in α-zein transcripts) would likely be needed. Introduction ofantisense genes and selection for reduced α-zein content and increasesin starch or other proteins of interest, would be followed byintroduction of these selected lines into a breeding program to optimizedesirable characteristics. It is believed that, in light of the presentdisclosure, one of skill in the art would now be able to alter thenutritional content of maize seeds through transformation of maizeplants using genes encoding antisense genes to the α-zeins.

Two α-zein antisense genes to the 19 kD zein (A20 gene) and 22 kD zein(Z4 gene) under control of the 10 kD zein promoter have been used totransform maize cells. These two plasmids were chosen from a variety ofplasmids containing antisense sequences to α-zeins based on reduction ofα-zein protein levels following hybrid arrest of α-zein RNA usingantisense transcripts, and in vitro translation of α-zein mRNA notremoved by hybridization. In these experiments, in vitro synthesizedsense and antisense RNAs were prepared and mixed using a 4:1 ratio ofantisense to sense RNA. Annealing conditions were determined by theappearance of sense::antisense hybrids on agarose gels. Although RNAswere successfully translated in both wheat germ and rabbit reticulocytesystems, the rabbit reticulocyte system was shown to be more efficient.Laser densitometry was used to quantitate the results of the in vitrotranslations. Several plasmids carrying antisense genes or parts ofantisense genes were examined, and the plasmids p1020p and pZ4ENT wereshown to be the most efficient in these assays.

Stable transformants were obtained by bombarding maize cells with theantisense constructs and selectable marker genes, and selection wascarried out as described elsewhere, using Basta, bialaphos orhygromycin. Presumptive transformed calli were screened for antisenseconstructs by PCR, using primers to the Z10 promoter and the nos 3'sequence. Seven lines were identified which were PCR-positive forpZ4ENT, 15 lines were PCR-positive for p1020P, and 1 line was positivefor both constructs. These lines were regenerated and crossed to variousinbred lines. Further transformation experiments using pDPG165 as theselectable marker gene, resulted in an additional 13 transformants whichcarried p1020P, 13 with pZ4ENT, and 42 which were PCR⁺ for bothplasmids. These transformed cell lines are now being regeneratedaccording to standard procedures.

R₁ seeds were collected from regenerated plants, and 110 R₁ seeds from 6antisense lines were grown in the greenhouse. Seed was uniform inappearance and synchronous germination occurred at 95%. Plants appearednormal, and selection for Basta resistance was carried out one monthafter planting by leaf painting with 2% Basta. Twenty four resistantplants were identified in this manner. These plants were selfed andcrossed to inbred lines. Immature kernels of selfed plants wereharvested at 10, 12 and 16 days after pollination and frozen in liquidnitrogen for future Northern analysis of antisense constructs.

3. Insect Resistance

The yield and efficiency of producing grain from maize throughout theworld is affected by the action of a number of insect pests. The insectpests that currently affect the US maize crop include: the European CornBorer (Ostrinia nubilalis;Hbn), Southwestern Corn Borer (Diatraeagrandiosella), Southern Cornstalk Borer (Diatraea crambidoides), LesserCornstalk Borer (Elasmopalpus lignosellus), the corn rootworm(Diabrotica spp.), the corn earworm (Heliothis zea), armyworms(Spodoptera frugiperda; Pseudaletia unipuncta), cutworms (e.g. blackcutworm: Agrotis ipsilon), wireworms, assorted grubs, Chinch Bugs(Blissus leucopterus), Corn Flea Beetles, Billbugs, Corn Root Aphids,Corn Leaf Aphids, Corn Planthopper. As well as directly affecting growthand yield, insect feeding can also lead to increased damage due toinfection by other pathogens, e.g. when the insect serves actively as avector for the pathogen (maize chlorotic mottle virus) or passively byopening the plant tissue to infection (stalk, root and ear rot fungi).Infection of the ear by fungi can also lead to unacceptible levels offungal toxins (aflatoxin) in maize grain. Furthermore, grain harvestedfrom maize can also be damaged in storage by a variety of insects (e.g.seed corn maggot, meal moths, worms, beetles and weevils).

The control of insect pests to prevent damage is achieved in the USmainly by adopting good integrated pest management (IPM) procedures thatinclude the use of certain farming (and storage) practices, the use ofchemical and biological control measures and the use of maize germplasmthat confers resistance or tolerance to insect pests. The use of some ofthese aspects of IPM are not always compatible with efficient,cost-effective farming or can detrimentally impact the environmentthrough the use of chemical insecticides. Also, while traditionalbreeding has produced resistance or tolerance to some insect pests, thelevel of resistance to several important insect pests as either beeninadequate to prevent economic levels of damage or is incompatible withmaintaining high yield. To circumvent these problems and to reduce theuse of chemical insecticide, it would be advantageous to introduceinsect resistance genes into maize from a variety of sources.

In the examples described below we have utilized the transformationprocess to introduce into maize plants two genes from diverse sourcesthat can, or have the potential to, control insect damage. We havedemonstrated expression and inheritance of the genes in maize and havealso demonstrated that the gene(s) can be used to confer resistance to amajor insect pest of maize, the European Corn Borer.

In the first example (see example 42), DNA coding for the endotoxin froma soil bacterium, Bacillus thuringiensis (Bt gene), and DNA coding forprotease inhibitor II protein from tomato (Lycopersicum esculentum) weresimultaneously transformed into maize cells and plants were regeneratedto produce fertile transgenic maize plants containing one or both of thegenes. In the second example (Example 43), a Bt gene alone wasintroduced. In both examples a selectable marker coding for resistanceto the herbicide bialaphos was also introduced and used to initiallyidentify transformants. The close genetic linkage of the herbicideresistance and insect resistance genes may provide some utility byallowing breeders to follow the inheritance of the insect resistancegenes by screening for herbicide resistance.

Potential insect resistance genes which can be introduced include theBacillus thuringiensis crystal toxin genes or Bt genes (Watrud et al.,1985). Preferred Bt toxin genes for use in such embodiments include theCryIA(b) and CryIA(c) genes (H. Hofte and H. R. Whiteley, 1989.Microbiol. Revs. 53: 242-255).

The poor expression of the prokaryotic Bt toxin genes in plants is awell-documented phenomenon, and the use of different promoters, fusionproteins, and leader sequences has not led to sufficient gene expressionto produce resistance in several plant species (Perlak et al, 1991). Wehave previously introduced expression vectors into maize that containthe native coding sequence for the HD73 Bt gene {a representative of thecryIA(C) class of Bt genes}. The gene was derived by cloning from the B.thuringiensis strain and modified by removing the genetic elementsnecessary for expression in its original host and replacing them withelements known to be capable of directing the initiation and terminationof transcription of other foreign genes in maize. The expression vectorproduced was transformed into regenerable maize cells that wereeventually regenerated into plants. The transformed maize cells andplants that were recovered that contained the Bt expression vectorfailed to provide resistance to ECB larvae.

To reduce this factor as an influence on Bt gene expression in maize, wehave synthesized new DNA sequences for the HD73 and HD1 Bt genes thatcode for the same amino acid sequences as their native counterparts butwhich replace codons that are rarely used in actively expressed maizegenes (less than 19% of the time) with codons that are most frequentlyused in highly expressed maize genes. The synthetic DNA sequencse codedfor the active portion of the Bt genes and contained approximately thefirst 613 codons of the Bt genes (including the f-met initiation codon;see FIGS. 1 and 2 for sequences). The HD73 Bt endotoxin gene wasintroduced into a plant expression vector similar to that previouslyused for the native Bt gene and also into expression vectors withmodified expression control elements designed to increase expression.Other examples of modified Bt toxin genes reported by others include thesynthetic Bt CryIA(b) gene and CryIA(c) genes (Perlak et al., 1991).

In the current examples, genes coding for protease inhibitors have alsobeen introduced into maize. The use of protease inhibitors to mediateresistance to insect pests has been described before (R. Johnson etal.,1989.; V. A. Hilder et al., 1987) but none of these genes havepreviously been reported to have been introduced into maize using thetransformation process. The use of a protease inhibitor II gene (PIN)from tomato or potato is envisioned to be particularly useful. Even moreadvantageous is the use of a PIN protein in combination with a Bt toxinprotein, the combined effect of which has been discovered by the presentinventors to produce synergistic insecticidal activity.

The two examples cited below illustrate the utility and benefits ofusing the current invention to introduce insect resistance genes intomaize.

EXAMPLE 42 Insect Resistance in Transgenic Plants

In this example AT824 cells were bombarded with 10 ug each pDPG165,pDPG354 and pDPG44 as described in example 10.

Transformants were selected similar to example 19. The bombarded cellswere removed from the microprojectile gun chamber, incubated on the wetfilters in the petri dish for 16-24 hours, scraped from the filter andplaced in liquid 409 medium (10 ml medium in a 125 ml conical flask).Flasks were incubated at ambient temperature (20-25° C.) in an orbitalshaker (New Brunswick Scientific, controlled environment, incubatorshaker; 125-150 rpm). Cells were subcultured (2 ml PCV into 20 mlmedium) every 3.5 days for one week without herbicide then subculturedin fresh 409 medium plus 1 mg/L bialaphos (medium 434) every 3.5 daysfor 2 weeks. Cells were then dispensed onto solid 425 medium (3 mg/Lbialaphos) at a density of 0.1 ml PCV per plate and incubated in thedark at ambient temperature. Approximately 250 plates were generated peroriginal bombarded filter and 3-5 weeks post plating colonies of cellspotentially resistant to the herbicide were identified.

Bialaphos resistant transformants were transferred to fresh solid 425medium and subcultured twice (once every two weeks) before selectedsamples were taken for analysis by polymerase chain reaction (PCR) todetect the presence of the Bt and/or protease inhibitor (PIN) genes

For PCR analysis, standard protocols were followed using the followingprimers:

PCR primers for PIN gene were:

PIN-1 (MD-1) (seq id no:18): 5'-GCT TAC CTA CTA ATT GTT CTT GG-3'

PIN-4: (MD-4) (seq id no:19): 5'-CAG GGT ACA TAT TTG CCT TGG G-3'

PCR primers for Bt gene were:

BTSN64 (seq id no:20): 5'-AAC CCT GAA TGG AAG TGC-3'

BTASN506 (seq id no:21): 5'-ACG GAC AGA TGC AGA TTG G-3'.

Forty-two of the putative transformants (Table 17) were analyzed by PCRfor the Bt gene and in most cases also for the PIN gene: A number werepositive for Bt or PIN alone and a majority of PCR positivetransformants were positive for both genes.

Regeneration of plants was similar to example 31. To regenerate thetransformed maize cells, the bialaphos resistant maize clones werepassaged (every two weeks; 24° C.) on the following media at 24° C.:

(i) On solid 223 medium or 425 medium and maintained for 1-10 passages(2-3 weeks per passage).

(ii) Passaged one to three times on 189 medium (first passage in thedark; later passages in low light; 16 hours light:8 hours dark),

(iii) Passaged one to four times on 101 medium in higher light.

(iv) Passaged one to four times on 607 or 501 medium in Plant Con®containers in higher light.

Once shoots were observed in tissue incubated on 101 medium, the lightintensity (fluorescent light: 25-250 mE·M.sup. 31 2·S⁻¹) was increasedby slowly adjusting the distance of the plates from the light sourcelocated overhead. Transformants that developed 3 leaves and 2-3 rootswere then transferred to a soilless plant growth mix. In some casesindolebutyric acid (3 ml of 0.3% w/v solution) was applied to the baseof rootless plants to stimulate root development.

Plantlets in soil were incubated in an illuminated growth chamber andconditions were slowly adjusted to adapt or condition the plantlets tothe drier and more illuminated conditions of the greenhouse. Afteradaptation/conditioning in the growth incubator, plants (R0 generation)were transplanted individually to large (5 gal) pots of soil andtransferred to the greenhouse.

Genotypic Analysis.

The genotype of several of the R0 plants was further analyzed bySouthern blot to determine if the transformants were independent and hadBt DNA inserted in variable locations within the maize genome. This wasachieved by: (i) cutting DNA isolated from the transformants with arestriction endonuclease (e.g. Kpn I). which cleaves to the 3' terminusof the Bt coding sequence and does not cleave the DNA sequence 5' to theBt gene in the vector and (ii) carrying out a Southern blot probing theDNA with a radioactively labeled DNA probe specific to the Bt gene. Thesize and number of the resulting bands detected by autoradiography wereindicative of the location of the nearest restriction endonuclease sitelocated in the maize genome 5' to the inserted Bt DNA sequence. Thesevaried in size for different independent insertion events. The resultsshowed that, for most of the transformants analyzed, the Bt DNA insertedinto different sites in the maize genome and most of the transformantswere independent transformants (i.e. not clonaly related to each other).

Demonstration of Expression of Bt Endotoxin and PIN Genes

Insect Bioassay (Resistance to European Corn Borer).

The regenerated (R0) plants were grown to early whorl stage (18"-26"extended leaf height with 5-6 leaves) and 5 infested with neonateEuropean corn borer larvae. A `Davis` inoculator (BIO-SERV, Frenchtown,N.J.) was used to reproducibly introduce a fixed number (80-120) ofnewly hatched European corn borer larvae(dispersed with corn cob grits.)into the whorl of the plants Following inoculation, the larvae wereallowed to feed on the plants for 2 weeks before they were evaluated forleaf damage. This infestation provided a simulation of an infestationwith first brood (first generation) European corn borer larvae.

As shown in Table 18, high levels of resistance to ECB were seen inseveral transformants containing the Bt gene. Since DNA, introduced intoplants by a variety of methods, can insert into a wide variety oflocations within the maize genome, the level of expression of anyinserted gene is variable and partly dependent on the location ofinsertion into the genome. The average level and pattern (developmentalor temporal) of expression is also dependent on the composition of theinserted DNA. This also appears to be the case for the current example,since while several of the individual transformants showed high levelsof resistance to insect feeding although a lower number containing theBt gene did not. Depending on the composition of the introduced DNA itwould usually be appropriate to evaluate a number of transformants toobtain transformants with the optimum level of expression of theintroduced DNA.

Demonstration of Transcription of Bt and Protease Inhibitor Genes

The transgenic R0 plants were further analyzed to determine whethertranscription (production of gene-specific RNA) of the introduced genescould be detected. Total RNA was isolated from leaf tissue excised fromthe transgenic maize plants and suitable negative controls and analyzedby northern blotting procedures. The RNA was first separated accordingto size by electrophoresis into an agarose gel (under denaturingconditions), transferred and bound to a membrane support and thenhybridized with gene specific, radio-labelled probes. The probes usedwere:

Bt gene: A fragment of DNA containing the first 1364 bp of the codingsequence of the synthetic Bt gene (approximately from the Nco I site atthe 5' terminus of the Bt gene to the Bam HI site 1364 bp into the Btgene).

Tom PIN gene: The Xba I-Bam HI fragment of pDPG344 containing the cDNAcoding sequence of the tomato protease inhibitor I gene.

barR gene: The Bam HI-Kpn I fragment from pDPG165 comprising the codingsequence of the barR gene.

After hybridization and washing to remove non-specifically bound probes,the membranes were exposed to X-ray film and analyzed. The appearance ofbands representing probe hybridizations to specific species of RNAdemonstrated the transcription of the introduced genes in the transgenicmaize plants. The presence of Bt RNA correlated well with the appearanceof resistance to the ECB larvae. In this case since expression of theTom PIN gene was also detected the contribution of this gene to theresistance could not be determined. Other transformants that areresistant and contain only the Bt gene, with no PIN gene present,indicate that the Bt gene can significantly increase the resistance ofmaize to ECB larvae (see Example 43 below).

Sexual Competency of R0 Transgenic Plants Containing Insect ResistanceGenes

The S23BI3602 and S23BI3702 R0 plants, containing the introduced insectresistance genes, were grown to sexual maturity and crossed tonon-transgenic inbred maize lines (e.g. inbreds "CN" and "AW") either byfertilizing the non-transgenic inbred maize plants with pollen derivedfrom transgenic maize or fertilizing the transgenic plants with pollenfrom non-transgenic inbreds. The techniques used to cross andself-pollinate transgenic maize plants were described in the CIP (filedApr. 11, 1990) of U.S. patent application Ser. No. 07/467983 (filed Jan.22, 1990). The yield of progeny was variable and depended on the inbredparent used, but sufficient seed was recovered to demonstrate that thetransgenic plants were fertile.

Sexual Transmission, Segregation and Expression of Insect ResistanceGenes in Progeny

The harvested seed (R1 generation) was harvested, planted in soil in 5gal pots and grown in the greenhouse. When the plants had grown so theyhad at least one true leaf extended, a 2%(w/v) solution of a commercialformulation of bialaphos (BASTA®, Hoechst TX100) was painted on a smallcircle of leaf tissue and after one week the plants were evaluated forherbicide resistance. Resistance to the herbicide was identified by thelack of a browning reaction (necrosis) in the area treated withherbicide (tissue browning=sensitivity, no browning=resistance).

Following this assay, samples were taken to determine the genotype ofthe segregants (by PCR assay) and the plants were infested with ECBlarvae to determine insect resistance phenotype. The results (Table 19)showed that the progeny inherited the Bt and PIN genes together with theresistance to bialaphos and ECB larvae. The low insect damage ratingnumber (high insect resistance) correlated with the presence of the Bt.No significant resistance to ECB larvae above the no-Bt controls wasever been detected in transgenic maize with only the BarR gene present.Furthermore, in a separate experiment when only the pDPG354 and pDPG165expression cassettes were maintained in the transformed cells, R0 and R1plants (transformant S25BJ18) the insect resistance phenotype was stillinherited, suggesting that the Bt gene (without PIN gene) is capable ofconfering resistance to ECB larvae.

There was no independent assortment of the introduced genes, indicatingthat the Bt, PIN and bialaphos resistance genes in transformant S23BI36or S23BI37 were closely linked. Resistance was inherited independent ofthe inbred used. Close linkage of the herbicide resistance and insectresistance will provide an advantage for the production of commercialmaize seed containing the insect resistance gene, since the presence ofthe Bt gene in subsequent generations can be detected and followed byscreening for herbicide resistance. This will allow for screening forthe Bt gene to take place in locations and times when infestation withinsects is impossible or difficult (e.g. in winter nurseries).

                  TABLE 17                                                        ______________________________________                                        PCR Analysis of transgenic maize cells (S23BI31 clones)                         containing Bt and/or Tom PIN genes.                                           Clone             Bt     PIN                                                  PIN PCR PCR                                                                 ______________________________________                                        01              --     +                                                        02 -- ND                                                                      03 -- ND                                                                      04 -- ND                                                                      05 + +                                                                        06 -- ND                                                                      07 + +                                                                        08 -- +                                                                       09 SG SG                                                                      10 + --                                                                       11 + +                                                                        12 + +                                                                        13 + +                                                                        14 + +                                                                        15 SG SG                                                                      16 + --                                                                       17 SG SG                                                                      18 -- ND                                                                      19 -- ND                                                                      20 -- ND                                                                      21 SG SG                                                                      22 + +                                                                        23 + +                                                                        24 + +                                                                        25 ND ND                                                                      26 -- ND                                                                      27 ND ND                                                                      28 -- --                                                                      29 + +                                                                        30 + +                                                                        31 + +                                                                        32 + +                                                                        33 + +                                                                        34 -- --                                                                      35 + --                                                                       36 + +                                                                        37 + +                                                                        38 + +                                                                        39 -- +                                                                       40 -- +                                                                       41 -- +                                                                       42 + +                                                                        43 + +                                                                        44 + +                                                                        45 + +                                                                        46 -- --                                                                      47 -- --                                                                      48 -- --                                                                    ______________________________________                                         Key:                                                                          + = target DNA present                                                        -- = target DNA not present                                                   ND = not determined                                                           SG =tissue stopped growing.                                              

                  TABLE 18                                                        ______________________________________                                        Expression of Bt gene in transgenic maize plants.                                            Bt       Resistance    PIN                                       R0 plant RNA Rating RNA RNA bar                                             ______________________________________                                        S23BI3015  +++      1        +      ++                                          S23BI3016 +++ 1 + ++                                                          S23BI3119 +++ 1 + ++                                                          S23BI3128 +++ 1 + ++                                                          S23BI3203 -- 5 -- +                                                           S23BI3202 -- 8 -- +                                                           S23BI3204 -- 8 -- +                                                           S23BI3704 +++ ND + ++                                                         523BI3708 +++ 2 ++ +++                                                        S23BI3709 +++ 1 ++ +++                                                        S23BI3712 +++ 1 ++ +++                                                        S23BI3715 +++ 1 ++ +++                                                        S23BI3716 +++ 2 ++ +                                                          S23BI3720 + 3 ND ND                                                           No-Bt control -- 5-9 -- -- --                                               ______________________________________                                         Key:                                                                          -- = no RNA detected resistant                                                + = low level Bt RNA susceptible                                              ++ = intermediate level of Bt RNA                                             +++ = higher level of Bt RNA                                                  ND = not determined                                                           Resistance ratings:                                                           1 = highly                                                                    9 = highly                                                               

                  TABLE 19                                                        ______________________________________                                        Inheritance and expression of insect resistance in                              progeny from Example 42.                                                      Transformant                                                                              Plant          PIN   BAR    ECB                                   {R:S RATIO} Number Bt PCR PCR resistance resistance                         ______________________________________                                        S23BI3602(CN)                                                                           02       --      --    S      6                                       {4R:65} 05 --  S 6                                                             07 ND ND S ND                                                                 08 + + R 1                                                                    09 -- -- S 6                                                                  10 + + R 1                                                                    11 ND ND S 9                                                                  13 -- -- S 3                                                                  14 + + R 1                                                                    15 + + R 1                                                                   S23BI3604(AW) 01 -- -- S 5                                                    {6R:9S} 02 + + R 1                                                             03 ND ND S 9                                                                  04 -- -- S 7                                                                  05 ND ND S ND                                                                 06 -- ND S 6                                                                  07 + + R 1                                                                    08 + + R 1                                                                    09 + + R 1                                                                    10 -- -- S 7                                                                  11 + + R 1                                                                    12 ND ND S 9                                                                  13 ND ND S 9                                                                  14 ND ND S ND                                                                 15 + + R 1                                                                   S23BI3702(bk) 01 ND ND S 9                                                    {6R:5S} 02 + ND R 1                                                            03 + + R 1                                                                    05 + + R 1                                                                    06 + + R 1                                                                    07 + + R 1                                                                    08 ND ND S 6                                                                  09 ND ND S 9                                                                  10 ND ND S 9                                                                  12 + ND R 1                                                                   13 -- -- S 6                                                                  15 ND ND S 9                                                                 S25BJ1801(AW) 05 + ND R 1                                                     {5R:2S} 08 + ND R 1                                                            10 + ND R 1                                                                   11 ND ND S 9                                                                  13 ND ND R 3                                                                  14 -- ND R 1                                                                  15 ND ND S 9                                                               ______________________________________                                         Key for Table 19:                                                             + = DNA present                                                               -- = DNA not present                                                          S = susceptible to bialaphos                                                  R = resistant to bialaphos                                                    ND =Not determined.                                                           ECB resistance:                                                               1 = Highly resistant                                                          9 = highly susceptible                                                   

EXAMPLE 43 Insect Resistant Transgenic Plants

Microprojectiles were coated with DNA as described in Example 10 except14 ul of pDPG165 and 14 ul of pDPG337 DNA were used. The bombarded cellswere transferred (on the filter) onto solid 409 medium and moistenedwith 0.5 ml of liquid 409 medium. Tissue was returned to liquid 401 pluscoconut water one day after bombardment. Selection in liquid 409 plus 1mg/L bialaphos (434 medium) began 8 days post-bombardment and the cellswere then treated as described in Example 42.

Genotype Analysis

The data obtained using the Bt PCR primers and techniques described inExample 42 above showed that 5 out of 7 clones tested contained the Btgene.

Regeneration of Transformants

Clones positive for Bt were subcultured on 425 medium for 2-5 months anddepending on the clone either:

(i) passaged on 409 solid medium (1st passage about 2 months and secondpassage about 2 weeks) or passaged on solid 223 medium for about 19daysbefore:

(ii) two passages on 189 solid medium (lst for about 14-19 days andsecond for about 10-14 days) followed by:

(iii) three passages on 101 solid media and;

(iv) one to two passages on 501 solid medium (about 2 weeks per passage)in Plant Con® containers.

Bioassay of R0 Plants

Clones developing shoots and roots on 501 medium in Plant Con®containers were transferred to a soilless mix, grown in the growth room,transferred to soil and the greenhouse and assayed for insect resistanceas described above. Two individual transformants, S18BF1102 andS18BF1401, were assayed for resistance to first brood ECB larvae andwere given ratings of 8 and 1, respectively. Since the average ratingfor the no-Bt controls was about 7, S18BF1401 (one R₀ plant from eachclone) was considered highly resistant and S18BF1102 was consideredsusceptible. Since the high resistance of clone S18BF1401 to ECB larvaehas not been seen for any regenerated maize plants unless they containeda Bt gene (with or without a PIN gene), we concluded that the resistancewas due to the expression of the introduced Bt gene.

Sexual Transmission, Segregation and Expression of Bt Gene in Progeny

The S18BF1401 plant was sexually crossed with inbred line "AW" and theharvested seed was germinated, grown and assayed for inheritance ofbialaphos resistance phenotype, Bt genotype and ECB resistancephenotype.

Seed harvested from R0 plant (R1 seed) was planted in soil in 5 galpots, grown in the greenhouse and assayed for resistance to bialaphosand ECB larvae as described above. Samples were taken to determine thegenotype of the segregants (by PCR assay). The results (Table 20) showthat the progeny inherited the bialaphos resistance and Bt genes andalso inherited the resistance to ECB larvae. The low insect damagerating number (high insect resistance) correlated with the presence ofthe Bt. There was no independent assortment of the selected (Bt) genesindicating that the Bt and bialaphos resistance genes in transformantS18BF1401 were closely linked.

These results show that the transformation process can be used tointroduce genes that confer resistance to insects and that the genes canbe inherited.

Examples of Other Expression Vectors

The structural gene or the genetic elements associated with theintroduced DNA are not limited to those described in the specificexamples mentioned above. We have introduced the pDPG354 vector with avector that carries the potato protease inhibitor I gene, as well as thebialaphos resistance gene. We have also obtained transformants thatcontain one or more of the following Bt expression cassettes:

(1) cassette which contains the promoter from the Adh I gene of maize,the intron I from the Adh I gene, the HD73 synthetic Bt (FIG. 12) genefollowed by a 3' sequence containing the poly A site from the nopalinesynthase (nos) gene of Agrobacterium tumefaceiens.

(2) cassette which contains the 35S promoter from CaMV, intron I fromAdh I, synthetic Bt gene (FIG. 12) followed by the nos poly A.

(3) cassette which contains the 35S promoter, Adh intron I, transitpeptide derived from the maize rbcs (RuBISCO) gene fused directly to theHD73 synthetic Bt gene (FIG. 12), followed by the nos poly A sequence.

(4) cassette which contains the 35S promoter, maize RuBISCO transitpeptide fused to the synthetic Bt gene (FIG. 12) such that the codonscoding for the first 8 amino acids the Bt protein are substituted withthe first 9 amino acids of the mature maize RuBISCO protein, followed bythe `transcript 7` 3' sequence (see Example 43).

In each case the transformants also contained the bar gene.

                  TABLE 20                                                        ______________________________________                                        Inheritance and expression of Bt gene in progeny of Example 43.                   Transgenic         Bt       Bt   ECB                                        Plant BarR DNA RNA Resistance                                               ______________________________________                                        03          R      +          +    3                                            05 R + + 3                                                                    07 R ND + 3                                                                   10 R + + 3                                                                    13 R + + 3                                                                    15 R + + 3                                                                    17 R + ND 3                                                                   01 S -- -- 9                                                                  02 S -- -- 9                                                                  04 S -- ND ND                                                                 06 S -- -- 9                                                                  08 S -- -- 9                                                                  09 S -- -- 6                                                                  11 S -- ND ND                                                                 12 S -- ND ND                                                                 14 S -- ND 9                                                                  16 S -- ND 8                                                                AT824 × AW (non-transformant controls)                                       1          S      ND       ND   8                                           2 S ND ND 9                                                                   3 S ND ND 9                                                                   4 S ND ND 9                                                                   5 S ND ND 9                                                                   6 S ND ND 9                                                                ______________________________________                                         Key:                                                                          R = Resistant to BASTA;                                                       S = Sensitive to bialaphos;                                                   ND = Not determined;                                                          + = detected;                                                                 -- = not detected                                                        

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes, which could be used alone or in combinationsinclude, for example:

1. Other Bt Genes.

Endotoxin genes from other species of Bacillus thuringiensis that aretoxic either affecting viability, growth or development of the pestinsects (Hofte, H. and Whitely, H. R., 1989).

2. Digestion Inhibitors.

Genes that code for inhibitors of the insect pest's digestive system.The gene products may themselves inhibit digestion or could code forenzymes or co-factors that facilitate the production of inhibitors: e.g.protease inhibitors such as the cowpea trypsin inhibitor (CPTI; V. A.Hilder et al.1987.) and oryzacystatin from rice (K. Abe et al., 1987)and amylase inhibitors such as amylase inhibitor from wheat and barley(J. Mundy and J. C. Rogers 1986). (For a reviews see C. A. Ryan. 1981.and C. A. Ryan, 1989).

3. Lectins.

Genes that control the production of lectins (T. H. Czapla and B. A.Lang. 1990; Chrispiels et al., 1991) such as wheat germ agglutinin(Raikhol, N. V. and Wilkins, T. A., 1987) that can affect the viabilityand/or growth of the insect pest.

4. Biological Peptides.

Genes controling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as (i) lyticpeptides (Westerhoff et al., 1989), (ii) peptide hormones (Kattaoka, H.et al., 1989; Keeley, L. L. and Hayes, T. K., 1987) and (iii) toxins andvenoms (Zlotkin, E., 1985; Dee, A. et al., 1990). Such polypeptidescould be synthesized in the plant as mono- or oligomers, as fusionproteins fused to carrier proteins, such as Bt, or delivered to theinsects in the presence of other agents, e.g. lectins, that maystimulate uptake or activity. Biological peptides may include peptidesdesigned by molecular modeling to interact with components in or oninsects to affect growth, development, viability and/or behaviour.

5. Lipoxygenases.

These naturally occuring plant enzymes have been shown to haveantinutirtional effects on insects and to reduce the nutritional qualityof their diet. Plants with enhanced lipoxygenase activity may beresistant to insect feeding (S. S. Duffey and G. W. Felton ,1991).

6. Production of Inadequate Nutrients or Removal of Essential Nutrients.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pests.Examples include: (i) genes that alter the sterol composition of plantsmay have a negative effect on insect growth and/or development and henceprovide the plant with insecticidal activity. Essential sterols could beconverted to undesirable sterols or undesirable sterols could beproduced directly; (ii) increasing levels or the characteristics ofpolyphenol oxidases and/or its substrate thereby increasing theproduction of toxic products or conversion of protein nutrients toundesirable by-products (Duffey, S. S. and Felton, G. W., 1991).

7. Qualitative or Quantitative Changes in Plant Secondary Metabolites.

DIMBOA: It has been demonstrated that some lines of maize showresistance to first brood European Corn borer larvae due to theproduction of DIMBOA (Klun, J. A. et al., 1967) in the plant. There arealso suggestions that DIMBOA production could have an impact on rootwormdamage. The introduction of DNA that changes quantity, timing or/andlocation of DIMBOA production may lead to improved resistance to severalmaize insect pests. Candidate genes for consideration would includethose involved in the pathway for production of DIMBOA, e.g. genes atthe bx locus (Dunn, G. M. et al., 1981)

Maysin: Since maysin has been implicated in the resistance of maize toearworm (Guildner, R. C., et al., 1991) the introduction of genes thatcan regulate the production of maysin may be beneficial to theproduction of insect resistant maize. Since the current invention allowsfor the transfer of genes from diverse sources into maize, the inventioncould also make possible the transfer of the capability to produce anyother secondary metabolite from any biological source into maizeprovided the genes needed to control the production of the metaboliteare available.

Dhurrin: genes involved in the production of dhurrin in sorghum(Branson, T. F., et al., 1969) which could be transferred to maize andfacilitate resistance to corn rootworms.

8. Genes Transferred from Native Grasses.

There are a number of native grasses that are resistant to some of theinsect pests of commercial inbred maize are susceptible to. Selectedlandraces of Tripsacum dactyloides have been reported to be resistant tocorn rootworms (Branson, T. F., 1971). Genes coding for the resistancetrait in this species could be isolated and transformed into maize toproduce rootworm resistant maize.

9. Cuticle Degradating Enzymes.

Genes that code for enzymes that affect the integrity of the insectcuticle such as chitinase, protease and lipase (M. S. Goettel et al.,1989; J. D. G. Jones et al., 1988) could be introduced into maize toproduce insect resistant plants. Including with these genes would begenes that code for activities that affect insect molting such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase (D. R.O'Reilly and L. K. Miller, 1989).

10. Antibiotics.

Genes that control the production of antibiotics that affect insectviability, growth or development, e.g. genes for the production ofnikkomycin--a compound that inhibits chitin synthesis (U. Schluter andG. Serfert, 1989) and avermectin and abamectin (Campbell, W. C., Ed.,1989; Ikeda et al., 1987) and insecticides from fungi (P. F. Dowd and O.K. Miller, 1990).

11. Antibodies.

Genes that can control the production of antibodies that can inhibitinsects. Antibodies have been produced in transgenic plants (Hiatt, A.et al., 1989). The genes coding for the antibodies to insect targetscould be cloned, engineered and transferred to maize using the currentinvention.

12. Pro-insecticides.

Another approach that can use genes introduced into maize is theintrodution of genes that code for enzymes that can covert a non-toxicinsecticide (pro-insecticide) applied to the outside of the plant intoan insecticide inside the plant. The benefits would be to: (i) reducethe levels of toxic insecticides applied to crops and (ii) make theinsecticide very selective since it would only be converted to theinsecticide inside the transgenic plants. Plants could further beengineered to degrade residual insecticide in plants parts destined forconsumption by introducing genes coding for degradative enzymes that areexpressed temporally or spacially to degrade thge insecticde in selectedtissues.

13. Others.

There are numerous other genes that have the potential to facilitateresistance to insects if introduced and expressed in maize. Theseinclude: ribosome inactivating proteins (A. M. R. Gatehouse et al.,1990)genes controlling expression of juvenile hormones (Hammock et al, 1990),genes that regulate plant structures (e.g. thickness of leaves andstalks, presence of trichomes, size of root system), the production ofchemicals that can deter insect pests, act as feeding deterrents orreduce the immunity of the insect pest to disease (D. W.Stanley-Samuelson et al.,1991).

In the above examples, depending on the source of the DNA, the DNA to beintroduced and expressed in maize may need to be modified to obtainoptimum expression (level, timing and location of expression) bychanging the genetic elements that regulate expression (promoters,introns, etc.) and the coding sequence (to improve translation, RNAstability and/or gene product activity).

The main element required for expression of an introduced gene is thestructural gene for the protein that mediates the resistance eitherdirectly as in the case of the insecticidal Bt protein, or indirectlysuch as in the case of an enzyme that might degrade nutrients essentialfor insect growth. In practice, most expression vectors will contain:(i) a promoter, located 5' to the coding sequence, to intiatetranscription of the introduced gene; (ii) the DNA sequence of the genecoding for the insect resistance factor and (iii) a sequence located 3'to the coding sequence to stimulate termination of transcription.Additional sequences (enhancers, introns, leader sequences, transit orsignal sequences and 3' elements (transcription terminators orpoly-adenylation sites) may be used to increase expression oraccummulation of the gene product.

Promoters:

Promoters that could be used include promoters from:

(a) the maize Adh I (Walker et al., 1987), cab (T. Sullivan et al.,1989.) rbcs (Lebrun et al.,1987), PEPCase (R. L. Hudspeth and J. W. Grula.1989) genes;

(b) genes that express in pollen (e.g., Hansen et al., 1989)

(c) genes isolated from tissue-specific libraries;

(d) any gene that is functional in maize;

(e) pathogens that replicate in plants, especially; monocotyledonousplants;

(f) synthetic promoters that utilize elements from various plant genesor gene from pathogens that replicate in plants;

(g) genes that are expressed in leaves, stalks, earshanks, collarsheaths or roots;

(h) genes that are expressed in leaves, stalks, earshanks. collarsheaths or roots but are not expressed in developing kernels.

In addition to promoters that produce adequate levels of the insectresistance gene product in the tissues eaten by the insect pest, it maysometimes be required to construct vectors that express the anti-sensemRNA of an insect resistance gene in the kernel, or other parts of theplant, in order to inhibit accummulation of the gene product inlocations where it may be undesirable.

Enhancers

Transcription enhancers or duplications of enhancers could be used toincrease expression. These enhancers are often found 5' to the start oftranscription in a pormoter that functions in eukaryotic cells, but canoften be inserted in the forward or reverse orientation 5' or 3' to thecoding sequence. Examples of enhancers include elements from the CamV35S promoter and octopine synthase genes (Ellis et al., 1987). and evenpromoters from non-plant enkayotes (e.g. yeast; J. MA et al.,1988 Nature334:631-633).

Leader sequences.

The DNA sequence between the transcription initiation site and the startof the coding sequence is termed the untranslated leader sequence. Theleader sequence can influence gene expression and compilations of leadersequences have been made to predict optimum or sub-optimum sequences andgenerate "consensus" and preferred leader sequences (C. P. Joshi, 1987).The sequences may increase or maintain mRNA stability and preventinappropriate initiation of translation. Sequences that are derived fromgenes that are highly expressed in plants, and in maize in particular,would be preferred. The leader sequence from the soybean rbcs (RuBISCO)gene was used in pDPG337.

Transit or Signal Peptides.

Sequences that are joined to the coding sequence of the resistance gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the outside of the cellularmembrane). By facilitating the transport of the protein intocompartments inside and outside the cell these sequences may increasethe accummulation of gene product protecting them from proteaolyticdegradation. These sequences also allow for additional mRNA sequencesfrom highly expressed genes to be attached to the coding sequence of theinsect resistance genes. Since mRNA being translated by ribosomes ismore stable than naked mRNA, the presence of translatable mRNA in frontof the gene may increase the overall stability of the mRNA transcriptfrom the insect resistance gene and thereby increase synthesis of thegene product. Since transit and signal sequences are usuallypost-translationally removed from the initail translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypepide.

In two examples (see section entitled "Examples of other expressionvectors." above) listed above, the transit peptide sequence for themaize RUBISCO gene was fused to the Bt gene and transformed intoregenerable maize cells.

3' Elements.

The most commonly used 3' elements include a sequence of DNA that actsas a signal to terminate transcription and allow for thepoly-adenylation of the 3' end of the mRNA coding for the gene product.These sequences can be obtained from a number of genes that aretranscribed in maize and often can be isolated from genes that expressedin other plants or pathogens that infect plants. The most commonly used3' elements are the 3' elements from: (i) the nopaline synthase genefrom Agrobacterium tumefasciens (M. Bevan et al., 1983. Nucleic AcidsRes. 11:369-385), (ii) the terminator for the T7 transcript from theoctopine synthase gene of Agrobacterium tumefasciens (and the 3' end ofthe protease inhibitor I or II genes from potato or tomato.

Conclusions:

The examples described above show that the invention can be used tointroduce insect resistance genes into maize and that functional genescan be successfully inherited. Thus the invention shows great utility inproducing transgenic maize plants of significant commercial value andbenefit, allowing for improved resistance to insect pests, improvedproductivity to the farmer and improved quality of the environment byreducing the dependency on chemical insecticides.

While the invention is susceptible to various modifications andalternative forms, specific embodiments thereof have been shown by wayof example in the drawings and herein be described in detail. It shouldbe understood, however, that it is not intended to limit the inventionto the particular forms disclosed, but on the contrary, the intention isto cover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

Abe, K. et al., (1987) J.Biol.Chem. 262: 16793-16797

Abel, P. P., Nelson, R. S., De, B., Hoffman, N., Rogers, S. G., Fraley,R. T. and Beachy, R. N. 1986. Science 232:738-743.

Adams, W. R., Adams, T. R., Wilston, H. M., Krueger, R. W., and Kausch,A. P., Silicone Capsules for Controlled Auxin Release, in preparation.

Adang, M. J. et al. (1985) Gene 289-300.

An, G. et al., (1989) Plant Cell 1: 115-122.

Armaleo et al. (1990) Current Genetics 17:97-103.

Armstrong C. L., Green C. E. (1985). Planta 164:207-214.

Armstrong, C. L., Green, C. E., and Phillips, R. L. 1991. Maize GenticsCoop Newsletter 65:92-93.

Barkai-Golan, R., Mirelman, D., Sharon, N. (1978) Arch. Microbiol116:119-124.

Barton, K. A., Whiteley, H. R. & Yang, K. S. (1987) Plant Physiol. 85,1103-1109.

Beck, E., Ludwig, G., Auerswald, E. A., Reiss, B., Schaller, H. 1982.Gene 19: 327-336.

Belanger and Kriz Genetics 129:863-872. 1991

Benfey, P. N., Ren, L. & Chua, N. H. (1989) EMBO Journal,8(8):2195-2202.

Berg, D. E., Egner, C., Hirschel, B. J., Howard, J., Jorgensen, R., andTisty, T. D. (1980) Cold Spring Harbor Symposium 45:448-465.

Bernal-Lugo, I. and Leopold, A. C. 1992. Plant Physiol. 98:1207-1210.

Berry-Lowe, S. L. et al.,1982. J.Mol.Appl.Genet 1:483-498

Bevan, M., 1984. Nucleic Acid Research 12: 8711-8721.

Bevan, M., Barnes, W. M., Chilton, M. D., 1983. Nucleic Acid Research.11:369-385.

Blackman, S. A., Obendorf, R. L., Leopold, A. C. 1992. Plant Physiol.100:225-230.

Bol, J. F., Linthorst, H. J. M., Cornelissen, B. J. C. 1990. Annu. Rev.Phytopath. 28:113-138.

Bottino P. J., (1975). Botany 15:1-16.

Bouchez D., Tokuhisa J. G., Llewellyn D. J., Dennis E. S. and Ellis J.G., (1989) EMBO Journal 8(13):4197-4204.

Bowler, C., Van Montagu, M., and Inze, D. 1992. Ann Rev. Plant Physiol.43:83-116.

Branson, T. F. et al., (1969) J. Econ.Entom. 62: 1375-1378.

Branson, T. F. (1971) Annals of Am. Soc of Entomol. 64:861-863.

Branson, T. F. and Guss, P. L. 1972. Proceedings North Central BranchEntomological Soceity of America 27:91-95.

Broakaert, W. F., Parijs, J., Leyns, F., Joos, H., Peumans, W. J. (1989)Science 245:1100-1102.

Buchanan-Wollaston, V., Snape, A., and Cannon, F. 1992. Plant CellReports 11:627-631.

Callis, J., Fromm, M., Walbot, V. (1987), Genes and Develop.1:1183-1200.

W. C. Campbell, ed. 1989. Avermectin and Abamectin.

Cao et al., Plant Cell Rep (1992) 11:586-591.

Carlsson J., Drevin H., Axen R. (1978). Biochem J. 173:723.

Cavener & Ray (1991). Nucl Acids Res 19:3185-3192.

Chauboute, H. E., Chambet, N., Philipps, G., Ehling, M. and Grigot, C.1987. Plant Mol. Biol. 8: 179-191.

Chaubet, N., Clement, B., Philipps, G. and Gigot, C. 1991. PlantMolecular Biology 17: 935-940.

Chandler, V. L., Radicella, J. P., Robbins, P. P., Chen, J., Turks, D.(1989), The Plant Cell 1:1175-1183

Chomet P. S., Wessler S., Dellaporta S. L. (1978). EMBO J 6:295-302.

Chrispiels, M. A. et al.,(1991) The Plant Cell 3:1-9

Chu C. C., Wang C. C., Sun C. S., Hsu C., Yin K. C., Chu C. Y., Bi F. Y.(1975). Scientia Sinica 18:659-668.

Clark, R. (1982). J. of Plant Nutrition 5:1039.

Coe, E. H., Neuffer, M. G., and Hoisington, D. A. (1988), in Corn andCorn Improvement, Sprague, G. F. and Dudley, J. W., eds., pp. 81-258

Comai, L., U.S. Pat. No. 4,535,060; and ATCC deposit 39256.

Comai L., Gacciotti D., Hiatt W. R., Thompson G., Rose R. E., Stalker D.(1985). Nature 317:741-744

Comai, L., Sen, L. C., Stalker, D. M. 1983. Science 221:370-371.

Conger, B. V., Novak, F. J., Afza, R., Erdelsky, K. (1987), Plant CellRep 6:345-347

Conkling, M. A., Cheng, C. L., Yamamoto, Y. T., Goodman, H. M. (1990),Plant Physiol. 93:1203-1211.

Coxson, D. S., McIntyre, D. D., and Vogel, H. J. 1992. Biotropica 24:121-133.

Cristou P., McCabe D. E., Swain W. F. (1988). Plant Physiol 87:671-674.

Cuozzo, M., O'Connell, K. M., Kaniewski, W., Fang, R. X., Chua, N. andTurner, N. 1988. Bio/Technology 6:549-553.

Cutler, A. J., Saleem, M., Kendell, E., Gusta, L. V., Georges, F.,Fletcher, G. L. (1989), J Plant Physiol 135:351-354.

Czapla and Lang (1990), J. Econ. Entomol. 83:2480-2485.

Davies, T. G. E., Thomas, H., Thomas, B., Rogers, L. J. 1990. PlantPhysiol. 93:588-595.

De Block, M., Botterman J., Vandiwiele M., Dockx J., Thoen C., GosseleV, Movva N. R., Thompson C., Van Montagu M., Leemans J. (1987). EMBO J.6:2513-2518; see also PCT Publication number WO 87/05629, published Sep.24, 1987.

De Block, M., Botterman J., Vandiwiele M., Dockx J., Thoen C., GosseleV, Movva N. R., Thompson C., Van Montagu M., Leemans J. (1989). PlantPhysiol 91:694-701.

Dee. A., R. M. Belagaje, K. Ward, E. Chio and Mei-Huei T. Lai (1990)BioTechnology 8:339-342.

Dekeyser R., Claes B., Marichal M., Van Montagu M., Caplan A. (1989).Plant Physiol 90:217-223.

Dekeyser et al. (1990), Plant Cell, 2:591-602.

Delannay X., LaVallee B. J., Proksch R. K., Fuchs R. L., Sims S. R.,Greenplate J. R., Marrone P. G., Dodson R. B., Augustine J. J., LaytonJ. G., Fischhoff D. A. (1989). Bio/Technol 7:1265-1269.

Dellaporta, S., Greenblatt, I., Kermicle, J., Hicks, J. B., Wessler, S.(1988) in Chromosome Structure and Function: Impact of New Concepts,18th Stadler Genetics Symposium, J. P. Gustafson and R. Appels, eds (NewYork: Plenum Press), pp. 263-282.

Dellaporta, S. L., Greenblatt, I. M., Kermicle, J., Hicks, J. B., andWessler, S. (1988), Stadler Symposium 11:263-282.

Depicker, A. G., Jacobs, A. M., and Van Montagu, M. C. 1988. Plant CellReports 7:63-66.

DeWet, J. R., Wood, K. V., DeLuca, M., Helinski, D. R., Subramani, S.1987. Mol. Cell Biol. 7:725-737.

Dhaese, P. et al.,1983, EMBO J. 2,419-426.

D'Halluin, K., Bonne, E., Bossut, M. De Beuckeleer, M., and Leemans, J.The Plant Cell 4: 1495-1505.

Doring, H. P. and Starlinger (1986), Ann. Rev. Genet. 20:175-200

Dowd, P. F. and O. K. Miller (1990) Entomol. exp. appl. 57: 23-28.

Duffey, S. S. and G. W. Felton ,1991. "Bioregulators," P. A. Hedin, Ed.,ACS Symp.Series #449).

Dunn, G. M. et al.,(1981) Can.J. Plant Sci., 61:583-593).

Dure, L., Crouch, M., Harada, J., Ho, T.-H. D., Mundy, J., Quatrano, R.,Thomas, T., and Sung, Z. R. 1989. Plant Molecular Biology 12:475-486.

Ebert, P. R., Ha, S. B., An. G. (1987), PNAS 84:5745-5749.

Eichholtz, D. A., Rogers, S. G., Horsch, R. B., Klee, H. J., Hayford,M., Hoffman, N. L., Bradford, S. B., Fink, C., Flick, J., O'Connell, K.M., Frayley, R. T. 1987. Somatic Cell Mol. Genet. 13: 67-76.

Ellis J. G., Llewellyn D. J., Walker J. C., Dennis E. S., and Peacock W.J., (1987) EMBO Journal 6(11):3203-3208.

Erdmann, N., Fulda, S., and Hagemann, M. 1992. J. Gen. Microbiology138:363-368.

European Patent Application 154,204 (9/11/85).

European Patent Application publication number 0218571 A2, publishedApr. 15, 1987.

Farrell & Beachy, (1990), Plant Mol. Biol. 15:821.

Federoff, N. (1989), "Maize Transposable Elements", in Mobile DNA. Wowe,M. M. and Berg, D. E., eds., Amer. Soc. Microbiol., Wash., D.C., pp.377-411.

Feinberg A. P., Vogelstein B. (1983). Anal Biochem 132:6-13.

Finkle B. J., Ulrich J. M., Rains W., Savarek S. J. (1985). Plant Sci42:133-140.

Fischhoff D. A., Bowdish K. S., Perlak F. J., Marrone P. G., McCormickS. M., Niedermeyer J. G., Dean D. A., Kusano-Kretzmer K., Mayer E. M.,Rochester D. E., Rogers S. G., Fraley R. T. Bio/Technol 5:807-813.

Fitzpatrick, T. 1993. Gen. Engineering News 22 (March 7): 7.

Fluhr, R., Moses, P., MOrelli, G., Coruzzi, C., Chua, N.-H. 1986. EMBOJ. 5: 2063-2071.

Fransz, P. F., de Ruijter, N. C. A., Schel, J. H. N. (1989), Plant CellRep 8:67-70

Frisch et al. Mol. Gen. Genet. 228:287-293, 1991

Fromm M. E., Taylor L. P., Walbot V. (1986). Nature 312:791-793.

Fromm, H., Katagiri, F., Chua, N. H. (1989), The Plant Cell 1:977-984.

Gallie, D. R., Lucas, W. J., Walbot, V. (1989), The Plant Cell1:301-311.

Gatehouse, A. M., Dewey, F. M., Dove, J., Fenton, K. A., Dusztai, A.(1984), J Sci Food Agric 35:373-380.

Gatehouse, A. M. R., Barbieri, L., Stirpe, F. and Croy, R. R. D. 1990.Entomol. Exp. Appl. 54:43-51.

Gelvin, S. B., Schilperoort, R. A., Varma, D. P. S., eds. PlantMolecular Biology Manual (1990).

Goettel, M. S., R. J. St. Leger, N. W. Rizzo, R. C. Staples, and D. W.Roberts (1989) J.Gen. Microbiol. 135:2233-2239.

Goff, S., Klein, T., Ruth, B., Fromm, M., Cone, K., Radicella, J.,Chandler, V. 1990. EMBO J.: 2517-2522. Graham, J. S., Hall, G., Pearce,G., Ryan, C. A. 1986 Mol. Cell. Biol. 2:1044-1051.

Graham, J. S. et al., 1985, J Biol Chem 260: 6561-6564

Guerrero, F. D., Jones, J. T., Mullet, J. E. 1990. Plant MolecularBiology 15: 11-26.

Guildner, R. C. et al., 1991, in "Naturally Occuring PestBioregulators." ed. P. A. Hedin. ACS Sympos. Series #449)

Gupta, A. S., Heinen, J. L., Holaday, A. S., Burke, J. J., and Allen, R.D. 1993. Proc. Natl. Acad. Sci USA 90:1629-1633.

Goring, D. R., Thomson, L., Rothstein, S. J. 1991. Proc. Natl. Acad Sci.USA 88:1770-1774.

Guilley, H. et al.,1982 Cell 30:763-773.

Hammock, B. D., Bonning, B. C., Possee, R. D., Hanzlik, T. N., andMaeda, S. (1990), Nature 344:458-461.

Hansen, D. D. et al 1989. Plant Cell 1:173-179

Haughn G. W., Smith J., Mazur B., Somerville, C. (1988). Mol Gen Genet211:266-271.

Hauptmann R. M., Vasil V., Ozias-Aikins P., Tabaeizadeh Z., Rogers S.G., Fraley R. T., Horsch R. B., Vasil I. K. (1988). Plant Physiol86:602-606.

Heidecker & Messing (1986). Ann Rev Plant Physiol, 37:439-466.

Hemenway, C., Fang, R., Kaniewski, W. K., Chua, N. and Turner, N. E.1988. The EMBO J. 7:1273-1280.

Hiatt. A., R. Caffetterkey and K. Bowdish (1989) Nature 342:76-78.

Hilder, V. A., Gatehouse, A. M. R., Sheerman, S. E., Barker, R. F.,Boulter, D. (1987) Nature 330:160-163.

Hinchee, M. A. W., Connor-Ward, D. V., Newell, C. A., McDonell, R. E.,Sato, S. J., Gasser, C. S., Fischhoff, D. A., Re, C. B., Fraley, R. T.,Horsch, R. B. (1988) Bio/technol 6:915-922.

Hofte, H. and Whiteley, H. R., 1989. Microbiological Reviews. 53:242-255.

Hudspeth , R. L. and J. W. Grula. 1989. Plant Mol. Biol. 12:579-589.

Ikuta, N., Souza, M. B. N., Valencia, F. F., Castro, M. E. B.,Schenberg, A. C. G., Pizzirani-Kleiner, A., Astolfi-Filho, S. (1990),Bio/technol 8:241-242.

Ikeda, H., Kotaki, H., Omura, S. (1987), J Bacteriol 169:5615-5621.

Ingelbrecht, I. L. W., Herman, L. M. F., Dekeyser, R. A., Van Montagu,M. C., Depicker, A. G. (1989), The Plant Cell 1:671-680.

IPRF European Patent Application No. 90033A.

Iturraga, G et al. (1989), Plant Cell 1:8447.

Jefferson R. A. (1987). P1 Mol Biol Repr 5:387-405.

Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. 1987. EMBO J. 6:3901-3907.

Jones, J. D. G., C. Dean, D. Gidoni, D. Gilbert, D. Bond-Nutter, R. Lee,J. Bedbrook, and P Dunsmuir. (1988) Mol. Gen. Genet. 212: 536-542.

Johnson, R., Norvdez, J., An, G, and Ryan, C. (1989), Proc. Natl. Acad.Sci. USA 86:9871-9875.

Joshi, C. P. (1987) Nucleic Acids Res., 15:6643-6653.

Kaasen, I., Falkenberg, P., Styrvold, O. B., Strom, A. R. 1992. J.Bacteriology 174:889-898.

Kaeppler et al. (1990) Plant Cell Reports 9: 415-418.

Karsten, U., West, J. A. and Zuccarello, G. 1992. Botanica Marina35:11-19.

Kattaoka, H., R. G. Troetschler, J. P. Li, S. J. Kramer, R. L. Carneyand D. A. Schooley. (1989) PNAS 86:2976-2980.

Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714.

Kay, R. et al.(1987) Science 236, 1299-1302

Keeley, L. L. and T. K. Hayes (1987). Insect Biochem 17:639-651.

Keller et al. (1989), EMBO J., 8(5):1309-1314.

Kirihara, J. A., Petri, J. B., and Messing, J. 1988. Gene 71:359-370,

Kirihara, J. A., Hunsperger, J. P., Mahoney, W. C., Messing, J. 1988.Mol. Gen. Genet. 211: 477-484.

Klein, T. M., Gradziel, T., Fromm, M. E., Sanford, J. C. 1988.Bio/Technology 6:559-563.

Klein T. M., Kornstein L., Sanford J. C., Fromm M. E. (1987). Nature327:70-73.

Klein T. M., Kornstein L., Sanford J. C., Fromm M. E. (1989). PlantPhysiol 91:440-444.

Klun, J. A. et al.,(1967) J. Econ. Entom. 60:1529-1533)

Koster, K. L. and Leopold, A. C. 1988. Plant Physiol. 88:829-832.

Kozak M. (1984). Nucl Acids Res 12:857-872.

Krzyzek, R., and Laursen, C. PCT Publication WO 92/12250.

Krzyzek et al. (1990), U.S. patent Application Ser. No. 07/635,279,filed Dec. 28, 1990.

Laemmli, U. K. 1970. Nature 227:280.

Laufs, J., Wirtz, U., Kammann, M., Matzeit, V., Schaefer, S., Schell,J., Czernilofsky, A. P., Baker, B., and Gronenborn, B. 1990. Proc. Natl.Acad. Sci: 7752-7756.

Laursen, C. M., Krzyzek, R. A., Flick, E. E., Anderson, P. C., Spencer,T. M. 1993. Plant Molecular Biology. Submitted.

Lawton, M. A., Tierney, M. A., Nakamura, I., Anderson, E., Komeda, Y.,Dube, P., Hoffman, N., Fraley, R. T., Beachy, R. N. (1987), Plant Mol.Biol. 9:315-324.

Lebrun, M., Waksman, G., and Freyssinet, G. 1987. Nucl. Acid. Res.15:4360

Lee et al. (1991), PNAS, 88:6389-6393.

Lee and Saier, 1983. J. of Bacteriol. 153-685.

Levings, C. S., III. 1990. Science 250: 942-947.

Lindsey and Jones (1987a), Planta, 172:346-355.

Lindsey and Jones (1987b), Plant Mol. Biol., 10:43-52.

Lindsey and Jones (1990), Physiol. Plant, 79:168-172.

Lorz H., Baker B., Schell J. (1985). Mol Gen Genet 199:178-182.

Loomis, S. H., Carpenter, J. F., Anchordoguy, T. J., Crowe, J. H., andBranchini, B. R. 1989. J. Expt. Zoology 252:9-15.

Lyznik L. A., Ryan R. D., Ritchie S. W., Hodges T. K. (1989). Plant MolBiol 13:151-16.

M A, J. et al.,1988 Nature 334:631-633).

Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning.Cold Spring Harbor Laboratory. First Ed.

Mariani, C., De Beuckeleer, M., Truettner, J., Leemans, J. and Goldberg,R. B. 1990. Nature 347:737-741.

McCabe D. E., Swain W. F., Martinell B. J., Cristou P. (1988).Bio/Technol 6:923-926.

McDaniel C. N., Poethig R. S. (198.8). Planta 175:13-22.

Mundy, J. and J. C. Rogers (1986) Planta 169:51-63

Mundy, J. and Chua, N.-H. 1988. The EMBO J. 7: 2279-2286.

Murdock et al., (1990) Phytochemistry 29:85-89.

Murakami T., Anzai H., Imai S., Satoh A., Nagaoka K., Thompson C. J.(1986). Mol Gen Genet 205:42-50.

Murashige T., Skoog F. (1962). Physiol Plant 15:473-497.

Napoli, C., Lenieux, C., Jorgense, R. 1990. Plant Cell 2:279-289.

Nelson R. S., McCormick S. M., Delannay X., Dube P., Layton J., AndersonE. J., Kaniewska M., Proksch R. K., Horsch R. B., Rogers S. G., FraleyR. T. Beachy R. N. (1988). Bio/Technol 6:403-409.

Nester, E. W. et al., (1984). Ann. Rev. Plant Physiol 35:387-413.

Odell, J. T., Nagy, F., Chua, N. H. (1985) Nature 313:810-812.

Ogawa, Y. et al (1973). Sci. Rep., Meija Seika 13:42-48.

Omirulleh, S, Abraham, M., Golovkin, M., Stefanov, I., Karabaev, M. K.,Mustardy, L., Morocz, S., Dudits, D. Plant Molecular Biology 21:415-428.

O'Reilly, D. R. and L. K. Miller (1989) Science 245: 1110-1112.

Ow, D. W., Wood, K. V., DeLuca, M., dewet, J. R., Helinski, D. R.,Howell, S. H. (1986) Science 234:856-859.

PCT No. WO 87/-00141.

Pearce, G., Strydom, D., Johnson, S., Ryan, C. A. 1991. Science 253:895-898.

Perlak F. J., Fuchs R. L., Dean D. A., McPherson S. L., and Fischhoff D.A., (1991) Proc. Natl. Acad. Sci. USA 88:3324-3328.

Pfahler P. L. (1967). Can J. Bot 45:836-845.

Phi-Van, L., Kries, J. P., Ostertag, W., Stratling, W. H. (1990), MolCell Biol 10:2302-2307.

Piatkowski, D., Schneider, K., Salamini, F. and Bartels, D. 1990. PlantPhysiol. 94:1682-1688.

Potrykus, I., Saul, M. W., Petruska, J., Paszkowski, J., Shillito, R. D.(1985), Mol Gen Genet 199:183-188

Potrykus I. (1989) Trends Biotechnol 7:269-273.

Prasher, et al. (1985) Biochem. Biophys. Res. Commun., 126(3):1259-1268.

Prioli L. M., Sondahl M. R. (1989). Bio/Technol 7:589-594.

Puite et al. (1985) Plant Cell Rep. 4:274-276.

Raikhol, N. V. and T. A. Wilkins, 1987, PNAS 84:6745-6749.

Reed, R. H., Richardson, D. L., Warr, S. R. C., Stewart, W. D. P. 1984.J. Gen. Microbiology 130:1-4.

Rhodes C. A., Pierce D. A., Mettler I. J., Mascarenhas D., Detmer J. J.(1988). Science 240:204-207.

Richaud, R., Richaud, C., Rafet, P. and Patte, J. C. 1986. J. Bacteriol.166: 297-300.

Ryan, C. A. (1981) Biochemistry of Plants 6: ch. 6, Acad.Press

Ryan, C. A. (1989) BioEssays 10:20-24.

Sambrook, J., Fritsch, E. F., and Maniatus, T. (1989), MolecularCloning, A Laboratory Manual 2nd ed.

Schluter, U. and Seifert, G. 1989. J. Inver. Pathol. 53:387-391.

Schmidt, R. J., Ketudat, M., Ankerman, M. J. and Hoschek, G. 1992. PlantCell 4: 689-700.

Shagan, T., Bar-Zvi, D. 1993. Plant Physiol. 101:1397-1398.

Shah D. M., Horsch R. B., Klee H. J., Kishore G. M., Winter J. A., TumerN. E., Hironaka C. M., Sanders P. R., Gasser C. S., Aykent S., Siegel N.R., Rogers S. G., Fraley R. T. (1986). Science 233:478-481.

Shapiro, J. A. (1983), Mobile Genetic Elements, Academic Press, N.Y.

Shillito R. D., Carswell G. K., Johnson C. M., DiMaio J. J., Harms C. T.(1989). Bio/Technol 7:581-587.

Shimamoto K., Terada R., Izawa T., Fujimoto H. (1989). Nature338:274-276.

Shure M., Wesler S., Federoff, N. (1983). Cell 35:225-233.

Skriver, K. and Mundy, J. 1990. Plant Cell 2:503-512.

Smith, J. J., Raikhel, N. V. 1989. Plant Mol. Biology 13: 601-603.

Smith, C. J. S., Watson, C. F., Bird, C. R., Ray, J., Schuch, W. andGrierson, D. 1990. Mol. Gen. Genet. 224:447-481.

Southern E. M. (1975). J Mol Biol 98:503-517.

Spencer, T. M., Gordon-Kamm, W., Daines, R., Start, W., Lemaux, P. 1990.Theor. Appl. Genet. 79: 625-631.

Spencer, T. M., O'Brien, J. V., Start, W. G., Adams, T. R., Gordon-Kamm,W. J. and Lemaux, P. G. 1992. Plant Molecular Biology 18:201-210.

Spencer, T. M., Laursen, C. M., Krzyzek, R. A., Anderson, P. C. andFlick, C. E. 1993. Proceedings of the NATO Advanced

Study Institute on Plant Molecular Biology. In press.

Stalker, D. M., McBride, K. E., and Malyj, L. Science 242: 419-422,1988.

Stalker, D. M., Malyj, L. D., McBride, K. E. (1988), J Biol Chem263:6310-6314.

Stanley-Samuelson. D. W., E. Jensen, K. W. Nickerson, K. Tiebel, C. L.Ogg and R. W. Howard (1991) PNAS 88: 1064-1068.

Stief, A., Winter, D., Stratling, W. H., and Sippel, A. E. (1989),Nature 341:343.

Stiefel et al. (1990), The Plant Cell, 2:785-793.

Stougaard, J. 1993. The Plant Journal 3:755-761.

Sullivan, T. et al.,1989. Mol. Gen. Genet 215:431-440)

Sutcliffe, J. G. (1978), Proc Natl Acad Sci USA 75:3737-3741.

Szoka, U.S. Pat. No. 4,394,448.

Tarczynski, M. C., Jensen, R. G., and Bohnert, H. J. (1992), Proc. Natl.Acad. Sci. USA, 89: 2600

Tarczynski, M. C., Jensen, R. G., and Bohnert, H. J. 1993. Science259:508-510.

Thillet, J., Absil, J., Stone, S. R., Pictet, R. (1988), J Biol Chem263:12500-12508.

Thompson C. K., Movva N. R., Tizard R., Crameri R., Davies J. E.,Lauwereys M., Botterman J. (1987). EMBO J 6:2519-2623.

Timmermans, M. C. P., Maliga, P., Maliga, P., Vieiera, J. and Messing,J. 1990. J. Biotechnol. 14: 333-344.

Tomes D. (1990). Annual Meting Proceedings, 26th Annual Corn BreedersSchool, University of Illinois, Feb. 26-27, pp. 7-9.

Topfer et al. (1989), Plant Cell, 1:133-139.

Topfer et al. (1990), Physiol. Plant, 79:158-162.

Twell D., Klein T. M., Fromm M. E., McCormick S. (1989). Plant Physiol91:1270-1274.

Ueda, T. and Messing, J. 1991. Theor. Appl. Genet. 82: 93-100.

Ugaki et al., (1991). Nucl Acid Res, 19:371-377. U.S. Pat. No.4,535,060.

Vaeck M., Reynaerts A., Hofte H., Jansens S., De Beuckeleer M., Dean C.,Zabeau M., Van Montagu M., Leemans J. (1987). Nature 328:33-37.

Vain, P., Yean, H., and Flament, P. 1989. Plant Cell, Tissue and OrganCulture 18: 143-151.

van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N. M. 1990. PlantCell 2:291-299.

van Rensburg, L., Kruger, G. H. J., and Kruger, H. 1993. J. PlantPhysiol. 141:188-194.

Vasil, V., Clancy, M., Ferl, R. J., Vasil, I. K., Hannah, L. C. (1989),Plant Physiol. 91:1575-1579.

Vernon, D. M. and Bohnert, H. J. 1992. The EMBO J.11:2077-2085.

Waldron, C., Murphy, E. B., Roberts, J. L., Gustafson, G. D., Armour, S.L., and Malcolm, S. K. Plant Molecular Biology 5: 103-108, 1985.

Walker, J. C., Howard, E. A., Dennis, E. S., Peacock, W. J (1987), PNAS84:6624-6628.

Walters, D. A., Vetsch, C. S., Potts, D. E., and Lundquist, R. C. PlantMol Biol 18:189-200, 1992.

Wang, Y., Klein, T., Fromm, M., Cao, J., Sanford, J., Wu, R. 1988. PlantMolecular Biology 11:433-439.

Wang, Y., Zhang, W., Cao, J., McEhoy, D. and Ray Wu. 1992. Molecular andCellular Biology 12: 3399-3406.

Watrud, L. S., Perlak, F. J., Tran, M.-T., Kusano, K., Mayer, E. J.,Miller-Widemann, M. A., Obukowicz, M. G., Nelson, D. R., Kreitinger, J.P., and Kaufman, R. J. (1985), in Engineered Organisms and theEnvironment, H. O. Halvorson et al., eds., Am. Soc. Microbiol.,Washington, D.C.

Westerhoff, H. V., D. Juretic, R. W.hendler and M. Zasloff. (1989) PNAS86: 6597-6601.

White, J., Chang, S. P., Bibb, M. J., Bibb, M. J. (1990), Nucl AcidsRes, 18:1062.

Withers L. A., King P. J. (1979). Plant Physiol 64:675-678.

Wolter, F., Schmidt, R., and Heinz, E. 1992. The EMBO J. 4685-4692.

Wong, Y. C. et al. (1988). Plant Mol Biol 11:433-439.

Wyn Jones, R. G., and Storey, R. 1981. Physiology and Biochemistry ofDrought Resistance in Plants:171-204.

Xiang, C. and Guerra, D. J. 1993. Plant Physiol. 102:287-293.

Yamaguchi-Shinozaki, K., Koizumi, M., Urao, S., Shinozaki, K. 1992.Plant Cell Physiol. 33:217-224.

Yang, N. S., Russell, D. (1990), PNAS 87:4144-4148.

Yang H., Zhang M. H., Davey M. R., Mulligan B. J., Cocking E. C. (1988).Plant Cell Rep 7:421-425.

Yang, L. Y., Gross, P. R., Chen, C. H., Lissis, M. 1992. Plant MolecularBiology 18: 1185-1187.

Yugari et al J. Biol. Chem 240:4710-4716, 1965.

Zhang M. H., Yang H., Rech E. L., Golds T. J., David A. S., Mulligan B.J., Cocking E. C., Davey E. R. (1988). Plant Cell Rep 7:379-384.

Zlotkin, E. 1985. Comprehensive Insect Physiol. Biochem. Parmacol. vol10, chapter 15:499-541

Zukowsky et al. (1983), Proc. Natl. Acad. Sci. USA 80:1101-1105.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 22                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Met Ala Thr Val Pro Glu Leu Asn Cys Glu Me - #t Pro Pro Ser Asp         1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - GAGGATCCGT CGACATGGTA AGCTTAGCGG GCCCC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GATCCGTCGA CCATGGCGCT TCAAGCTTC         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - GCAGCTGGTA CCGCGAAGTT CGAAGGGCT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1845 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1839                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC AT - #T CCT TAC AAT TGC CTC           48                                                                       Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu             1               5 - #                 10 - #                 15              - - AGC AAC CCT GAA GTG GAA GTG CTG GGT GGC GA - #A CGC ATC GAA ACC GGT           96                                                                       Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly                        20     - #             25     - #             30                  - - TAC ACC CCA ATC GAT ATT TCC CTG TCC CTG AC - #C CAA TTT CTG CTG AGC          144                                                                       Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser                    35         - #         40         - #         45                      - - GAA TTT GTG CCC GGT GCT GGC TTT GTG CTG GG - #C CTG GTG GAT ATC ATC          192                                                                       Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile                50             - #     55             - #     60                          - - TGG GGC ATT TTT GGT CCC TCC CAA TGG GAC GC - #C TTT CTG GTG CAA ATT          240                                                                       Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile            65                 - # 70                 - # 75                 - # 80       - - GAA CAG CTG ATT AAC CAA CGC ATC GAA GAA TT - #C GCT AGG AAC CAA GCC          288                                                                       Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala                            85 - #                 90 - #                 95              - - ATT TCC CGC CTG GAA GGC CTG AGC AAT CTG TA - #C CAA ATT TAC GCC GAA          336                                                                       Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu                       100      - #           105      - #           110                  - - TCC TTT CGC GAG TGG GAA GCC GAT CCT ACC AA - #T CCA GCC CTG CGC GAA          384                                                                       Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu                   115          - #       120          - #       125                      - - GAG ATG CGC ATT CAA TTC AAT GAC ATG AAC AG - #C GCC CTG ACC ACC GCT          432                                                                       Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala               130              - #   135              - #   140                          - - ATT CCT CTG TTT GCC GTG CAA AAT TAC CAA GT - #G CCT CTG CTG TCC GTG          480                                                                       Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TAC GTG CAA GCT GCC AAT CTG CAT CTG TCC GT - #G CTG CGC GAT GTG        TCC      528                                                                    Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser                          165  - #               170  - #               175              - - GTG TTT GGC CAA AGG TGG GGC TTT GAT GCC GC - #C ACC ATC AAT AGC CGC          576                                                                       Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg                       180      - #           185      - #           190                  - - TAC AAT GAT CTG ACC AGG CTG ATT GGC AAC TA - #C ACC GAT TAC GCT GTG          624                                                                       Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp Tyr Ala Val                   195          - #       200          - #       205                      - - CGC TGG TAC AAT ACC GGC CTG GAA CGC GTG TG - #G GGC CCA GAT TCC CGC          672                                                                       Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg               210              - #   215              - #   220                          - - GAT TGG GTG AGG TAC AAT CAA TTT CGC CGC GA - #A CTG ACC CTG ACC GTG          720                                                                       Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - CTC GAT ATC GTG GCT CTG TTC CCA AAT TAC GA - #T AGC CGC CGC TAC        CCA      768                                                                    Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As - #p Ser Arg Arg Tyr Pro                          245  - #               250  - #               255              - - ATT CGA ACC GTG TCC CAA CTG ACC CGC GAA AT - #T TAC ACC AAC CCA GTG          816                                                                       Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val                       260      - #           265      - #           270                  - - CTG GAA AAT TTT GAT GGT AGC TTT CGC GGC TC - #C GCT CAG GGC ATC GAA          864                                                                       Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu                   275          - #       280          - #       285                      - - CGC AGC ATT AGG AGC CCA CAT CTG ATG GAT AT - #C CTG AAC AGC ATC ACC          912                                                                       Arg Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr               290              - #   295              - #   300                          - - ATC TAC ACC GAT GCT CAT AGG GGT TAC TAC TA - #C TGG TCC GGC CAT CAA          960                                                                       Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty - #r Trp Ser Gly His Gln           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ATC ATG GCT TCC CCT GTG GGC TTT TCC GGG CC - #A GAA TTC ACC TTT        CCA     1008                                                                    Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro                          325  - #               330  - #               335              - - CTG TAC GGC ACG ATG GGC AAT GCC GCT CCA CA - #A CAA CGC ATT GTG GCT         1056                                                                       Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala                       340      - #           345      - #           350                  - - CAA CTG GGT CAG GGC GTG TAC CGC ACC CTG TC - #C TCC ACC CTG TAC CGC         1104                                                                       Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg                   355          - #       360          - #       365                      - - CGC CCT TTT AAT ATC GGC ATC AAC AAC CAG CA - #A CTG TCC GTG CTG GAC         1152                                                                       Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp               370              - #   375              - #   380                          - - GGC ACC GAA TTT GCT TAC GGC ACC TCC TCC AA - #T CTG CCA TCC GCT GTA         1200                                                                       Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - TAC CGC AAG AGC GGC ACC GTG GAT TCC CTG GA - #T GAA ATC CCA CCA        CAG     1248                                                                    Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln                          405  - #               410  - #               415              - - AAT AAC AAC GTG CCA CCT AGG CAA GGC TTT AG - #C CAT CGC CTG AGC CAT         1296                                                                       Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His                       420      - #           425      - #           430                  - - GTG TCC ATG TTT CGC TCC GGC TTT AGC AAT AG - #C AGC GTG AGC ATC ATC         1344                                                                       Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile                   435          - #       440          - #       445                      - - CGC GCT CCT ATG TTC TCC TGG ATC CAT CGC AG - #C GCT GAA TTT AAC AAC         1392                                                                       Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn               450              - #   455              - #   460                          - - ATC ATT GCC TCC GAT AGC ATT ACC CAA ATC CC - #T GCC GTG AAG GGC AAC         1440                                                                       Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pr - #o Ala Val Lys Gly Asn           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - TTT CTG TTT AAT GGT TCC GTG ATT TCC GGC CC - #A GGC TTT ACC GGT        GGC     1488                                                                    Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pr - #o Gly Phe Thr Gly Gly                          485  - #               490  - #               495              - - GAC CTG GTG CGC CTG AAT AGC AGC GGC AAT AA - #C ATT CAG AAT CGC GGC         1536                                                                       Asp Leu Val Arg Leu Asn Ser Ser Gly Asn As - #n Ile Gln Asn Arg Gly                       500      - #           505      - #           510                  - - TAC ATT GAA GTG CCA ATT CAC TTC CCA TCC AC - #C TCC ACC CGC TAC CGC         1584                                                                       Tyr Ile Glu Val Pro Ile His Phe Pro Ser Th - #r Ser Thr Arg Tyr Arg                   515          - #       520          - #       525                      - - GTG CGC GTG CGC TAC GCT TCC GTG ACC CCA AT - #T CAC CTC AAC GTT AAC         1632                                                                       Val Arg Val Arg Tyr Ala Ser Val Thr Pro Il - #e His Leu Asn Val Asn               530              - #   535              - #   540                          - - TGG GGC AAT TCC TCC ATT TTT TCC AAT ACC GT - #G CCA GCT ACC GCT ACC         1680                                                                       Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Va - #l Pro Ala Thr Ala Thr           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - TCC CTG GAT AAT CTG CAA TCC AGC GAT TTT GG - #T TAC TTT GAA AGC        GCC     1728                                                                    Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gl - #y Tyr Phe Glu Ser Ala                          565  - #               570  - #               575              - - AAT GCT TTT ACC TCC TCC CTG GGT AAT ATC GT - #G GGT GTG CGC AAT TTT         1776                                                                       Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Va - #l Gly Val Arg Asn Phe                       580      - #           585      - #           590                  - - AGC GGC ACC GCC GGC GTG ATC ATC GAC CGC TT - #T GAA TTT ATT CCA GTG         1824                                                                       Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Ph - #e Glu Phe Ile Pro Val                   595          - #       600          - #       605                      - - ACC GCC ACC CTC GAG TAGGTA        - #                  - #                    1845                                                                     Thr Ala Thr Leu Glu                                                               610                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 613 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu        1               5 - #                 10 - #                 15              - - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly                   20     - #             25     - #             30                  - - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser               35         - #         40         - #         45                      - - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           50             - #     55             - #     60                          - - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile       65                 - # 70                 - # 75                 - # 80       - - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala                       85 - #                 90 - #                 95              - - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu                  100      - #           105      - #           110                  - - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu              115          - #       120          - #       125                      - - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala          130              - #   135              - #   140                          - - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val        Ser                                                                                             165  - #               170  - #               175             - - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg                  180      - #           185      - #           190                  - - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp Tyr Ala Val              195          - #       200          - #       205                      - - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg          210              - #   215              - #   220                          - - Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As - #p Ser Arg Arg Tyr        Pro                                                                                             245  - #               250  - #               255             - - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val                  260      - #           265      - #           270                  - - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu              275          - #       280          - #       285                      - - Arg Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr          290              - #   295              - #   300                          - - Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty - #r Trp Ser Gly His Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe        Pro                                                                                             325  - #               330  - #               335             - - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala                  340      - #           345      - #           350                  - - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg              355          - #       360          - #       365                      - - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp          370              - #   375              - #   380                          - - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro        Gln                                                                                             405  - #               410  - #               415             - - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His                  420      - #           425      - #           430                  - - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile              435          - #       440          - #       445                      - - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn          450              - #   455              - #   460                          - - Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pr - #o Ala Val Lys Gly Asn      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pr - #o Gly Phe Thr Gly        Gly                                                                                             485  - #               490  - #               495             - - Asp Leu Val Arg Leu Asn Ser Ser Gly Asn As - #n Ile Gln Asn Arg Gly                  500      - #           505      - #           510                  - - Tyr Ile Glu Val Pro Ile His Phe Pro Ser Th - #r Ser Thr Arg Tyr Arg              515          - #       520          - #       525                      - - Val Arg Val Arg Tyr Ala Ser Val Thr Pro Il - #e His Leu Asn Val Asn          530              - #   535              - #   540                          - - Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Va - #l Pro Ala Thr Ala Thr      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gl - #y Tyr Phe Glu Ser        Ala                                                                                             565  - #               570  - #               575             - - Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Va - #l Gly Val Arg Asn Phe                  580      - #           585      - #           590                  - - Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Ph - #e Glu Phe Ile Pro Val              595          - #       600          - #       605                      - - Thr Ala Thr Leu Glu                                                          610                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1848 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1842                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - ATG GAT AAC AAT CCG AAC ATC AAT GAA TGC AT - #T CCT TAC AAT TGC CTC           48                                                                       Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu             1               5 - #                 10 - #                 15              - - AGC AAC CCT GAA GTG GAA GTG CTG GGT GGC GA - #A CGC ATC GAA ACC GGT           96                                                                       Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly                        20     - #             25     - #             30                  - - TAC ACC CCA ATC GAT ATT TCC CTG TCC CTG AC - #C CAA TTT CTG CTG AGC          144                                                                       Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser                    35         - #         40         - #         45                      - - GAA TTT GTG CCC GGT GCT GGC TTT GTG CTG GG - #C CTG GTG GAT ATC ATC          192                                                                       Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile                50             - #     55             - #     60                          - - TGG GGC ATT TTT GGT CCC TCC CAA TGG GAC GC - #C TTT CTG GTG CAA ATT          240                                                                       Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile            65                 - # 70                 - # 75                 - # 80       - - GAA CAG CTG ATT AAC CAA CGC ATC GAA GAA TT - #C GCT AGG AAC CAA GCC          288                                                                       Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala                            85 - #                 90 - #                 95              - - ATT TCC CGC CTG GAA GGC CTG AGC AAT CTG TA - #C CAA ATT TAC GCC GAA          336                                                                       Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu                       100      - #           105      - #           110                  - - TCC TTT CGC GAG TGG GAA GCC GAT CCT ACC AA - #T CCA GCC CTG CGC GAA          384                                                                       Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu                   115          - #       120          - #       125                      - - GAG ATG CGC ATT CAA TTC AAT GAC ATG AAC AG - #C GCC CTG ACC ACC GCT          432                                                                       Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala               130              - #   135              - #   140                          - - ATT CCT CTG TTT GCC GTG CAA AAT TAC CAA GT - #G CCT CTG CTG TCC GTG          480                                                                       Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val           145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - TAC GTG CAA GCT GCC AAT CTG CAT CTG TCC GT - #G CTG CGC GAT GTG        TCC      528                                                                    Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val Ser                          165  - #               170  - #               175              - - GTG TTT GGC CAA AGG TGG GGC TTT GAT GCC GC - #C ACC ATC AAT AGC CGC          576                                                                       Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg                       180      - #           185      - #           190                  - - TAC AAT GAT CTG ACC AGG CTG ATT GGC AAC TA - #C ACC GAT TAC GCT GTG          624                                                                       Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp Tyr Ala Val                   195          - #       200          - #       205                      - - CGC TGG TAC AAT ACC GGC CTG GAA CGC GTG TG - #G GGC CCA GAT TCC CGC          672                                                                       Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg               210              - #   215              - #   220                          - - GAT TGG GTG AGG TAC AAT CAA TTT CGC CGC GA - #A CTG ACC CTG ACC GTG          720                                                                       Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val           225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - CTC GAT ATC GTG GCT CTG TTC CCA AAT TAC GA - #T AGC CGC CGC TAC        CCA      768                                                                    Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As - #p Ser Arg Arg Tyr Pro                          245  - #               250  - #               255              - - ATT CGA ACC GTG TCC CAA CTG ACC CGC GAA AT - #T TAC ACC AAC CCA GTG          816                                                                       Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val                       260      - #           265      - #           270                  - - CTG GAA AAT TTT GAT GGT AGC TTT CGC GGC TC - #C GCT CAG GGC ATC GAA          864                                                                       Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu                   275          - #       280          - #       285                      - - CGC AGC ATT AGG AGC CCA CAT CTG ATG GAT AT - #C CTG AAC AGC ATC ACC          912                                                                       Arg Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr               290              - #   295              - #   300                          - - ATC TAC ACC GAT GCT CAT AGG GGT TAC TAC TA - #C TGG TCC GGC CAT CAA          960                                                                       Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty - #r Trp Ser Gly His Gln           305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - ATC ATG GCT TCC CCT GTG GGC TTT TCC GGG CC - #A GAA TTC ACC TTT        CCA     1008                                                                    Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe Pro                          325  - #               330  - #               335              - - CTG TAC GGC ACG ATG GGC AAT GCC GCT CCA CA - #A CAA CGC ATT GTG GCT         1056                                                                       Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala                       340      - #           345      - #           350                  - - CAA CTG GGT CAG GGC GTG TAC CGC ACC CTG TC - #C TCC ACC CTG TAC CGC         1104                                                                       Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg                   355          - #       360          - #       365                      - - CGC CCT TTT AAT ATC GGC ATC AAC AAC CAG CA - #A CTG TCC GTG CTG GAC         1152                                                                       Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp               370              - #   375              - #   380                          - - GGC ACC GAA TTT GCT TAC GGC ACC TCC TCC AA - #T CTG CCA TCC GCT GTA         1200                                                                       Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val           385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - TAC CGC AAG AGC GGC ACC GTG GAT TCC CTG GA - #T GAA ATC CCA CCA        CAG     1248                                                                    Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro Gln                          405  - #               410  - #               415              - - AAT AAC AAC GTG CCA CCT AGG CAA GGC TTT AG - #C CAT CGC CTG AGC CAT         1296                                                                       Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His                       420      - #           425      - #           430                  - - GTG TCC ATG TTT CGC TCC GGC TTT AGC AAT AG - #C AGC GTG AGC ATC ATC         1344                                                                       Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile                   435          - #       440          - #       445                      - - CGC GCT CCT ATG TTC TCC TGG ATC CAC CGC TC - #C GCT GAG TTC AAC AAC         1392                                                                       Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn               450              - #   455              - #   460                          - - ATC ATC CCG TCC TCC CAA ATC ACC CAA ATC CC - #G CTC ACC AAG TCC ACG         1440                                                                       Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr           465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - AAC CTC GGC TCC GGC ACG TCC GTC GTC AAG GG - #C CCG GGC TTC ACC        GGC     1488                                                                    Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr Gly                          485  - #               490  - #               495              - - GGC GAC ATC CTC CGC CGC ACG TCC CCG GGC CA - #G ATC TCC ACC CTC CGC         1536                                                                       Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg                       500      - #           505      - #           510                  - - GTC AAC ATC ACG GCT CCG CTG AGC CAG CGC TA - #C AGG GTG CGC ATC AGA         1584                                                                       Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg                   515          - #       520          - #       525                      - - TAC GCT AGC ACG ACC AAC CTG CAA TTC CAC AC - #G TCC ATC GAC GGC AGA         1632                                                                       Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg               530              - #   535              - #   540                          - - CCG ATC AAC CAG GGC AAC TTC AGC GCG ACG AT - #G AGC TCC GGG TCC AAC         1680                                                                       Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn           545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - CTC CAG TCC GGC TCC TTC CGC ACG GTC GGT TT - #C ACC ACG CCG TTC        AAC     1728                                                                    Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe Asn                          565  - #               570  - #               575              - - TTC TCC AAC GGC TCC TCC GTC TTC ACG CTC TC - #C GCT CAC GTC TTC AAC         1776                                                                       Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn                       580      - #           585      - #           590                  - - TCC GGC AAC GAG GTG TAC ATC GAC CGC ATC GA - #G TTC GTC CCG GCC GAG         1824                                                                       Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu                   595          - #       600          - #       605                      - - GTC ACC TTC GAG CTC GAG TAGGTA      - #                  - #                  1848                                                                     Val Thr Phe Glu Leu Glu                                                           610                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 614 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Il - #e Pro Tyr Asn Cys Leu        1               5 - #                 10 - #                 15              - - Ser Asn Pro Glu Val Glu Val Leu Gly Gly Gl - #u Arg Ile Glu Thr Gly                   20     - #             25     - #             30                  - - Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Th - #r Gln Phe Leu Leu Ser               35         - #         40         - #         45                      - - Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gl - #y Leu Val Asp Ile Ile           50             - #     55             - #     60                          - - Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Al - #a Phe Leu Val Gln Ile       65                 - # 70                 - # 75                 - # 80       - - Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Ph - #e Ala Arg Asn Gln Ala                       85 - #                 90 - #                 95              - - Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Ty - #r Gln Ile Tyr Ala Glu                  100      - #           105      - #           110                  - - Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr As - #n Pro Ala Leu Arg Glu              115          - #       120          - #       125                      - - Glu Met Arg Ile Gln Phe Asn Asp Met Asn Se - #r Ala Leu Thr Thr Ala          130              - #   135              - #   140                          - - Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Va - #l Pro Leu Leu Ser Val      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Tyr Val Gln Ala Ala Asn Leu His Leu Ser Va - #l Leu Arg Asp Val        Ser                                                                                             165  - #               170  - #               175             - - Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Al - #a Thr Ile Asn Ser Arg                  180      - #           185      - #           190                  - - Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Ty - #r Thr Asp Tyr Ala Val              195          - #       200          - #       205                      - - Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Tr - #p Gly Pro Asp Ser Arg          210              - #   215              - #   220                          - - Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Gl - #u Leu Thr Leu Thr Val      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Asp Ile Val Ala Leu Phe Pro Asn Tyr As - #p Ser Arg Arg Tyr        Pro                                                                                             245  - #               250  - #               255             - - Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Il - #e Tyr Thr Asn Pro Val                  260      - #           265      - #           270                  - - Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Se - #r Ala Gln Gly Ile Glu              275          - #       280          - #       285                      - - Arg Ser Ile Arg Ser Pro His Leu Met Asp Il - #e Leu Asn Ser Ile Thr          290              - #   295              - #   300                          - - Ile Tyr Thr Asp Ala His Arg Gly Tyr Tyr Ty - #r Trp Ser Gly His Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pr - #o Glu Phe Thr Phe        Pro                                                                                             325  - #               330  - #               335             - - Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gl - #n Gln Arg Ile Val Ala                  340      - #           345      - #           350                  - - Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Se - #r Ser Thr Leu Tyr Arg              355          - #       360          - #       365                      - - Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gl - #n Leu Ser Val Leu Asp          370              - #   375              - #   380                          - - Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser As - #n Leu Pro Ser Ala Val      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu As - #p Glu Ile Pro Pro        Gln                                                                                             405  - #               410  - #               415             - - Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Se - #r His Arg Leu Ser His                  420      - #           425      - #           430                  - - Val Ser Met Phe Arg Ser Gly Phe Ser Asn Se - #r Ser Val Ser Ile Ile              435          - #       440          - #       445                      - - Arg Ala Pro Met Phe Ser Trp Ile His Arg Se - #r Ala Glu Phe Asn Asn          450              - #   455              - #   460                          - - Ile Ile Pro Ser Ser Gln Ile Thr Gln Ile Pr - #o Leu Thr Lys Ser Thr      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asn Leu Gly Ser Gly Thr Ser Val Val Lys Gl - #y Pro Gly Phe Thr        Gly                                                                                             485  - #               490  - #               495             - - Gly Asp Ile Leu Arg Arg Thr Ser Pro Gly Gl - #n Ile Ser Thr Leu Arg                  500      - #           505      - #           510                  - - Val Asn Ile Thr Ala Pro Leu Ser Gln Arg Ty - #r Arg Val Arg Ile Arg              515          - #       520          - #       525                      - - Tyr Ala Ser Thr Thr Asn Leu Gln Phe His Th - #r Ser Ile Asp Gly Arg          530              - #   535              - #   540                          - - Pro Ile Asn Gln Gly Asn Phe Ser Ala Thr Me - #t Ser Ser Gly Ser Asn      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Leu Gln Ser Gly Ser Phe Arg Thr Val Gly Ph - #e Thr Thr Pro Phe        Asn                                                                                             565  - #               570  - #               575             - - Phe Ser Asn Gly Ser Ser Val Phe Thr Leu Se - #r Ala His Val Phe Asn                  580      - #           585      - #           590                  - - Ser Gly Asn Glu Val Tyr Ile Asp Arg Ile Gl - #u Phe Val Pro Ala Glu              595          - #       600          - #       605                      - - Val Thr Phe Glu Leu Glu                                                      610                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 195 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AGCTTGCAGC GAGTACATAC ATACTAGGCA GCCAGGCAGC CATGGCGCCC AC -             #CGTGATGA     60                                                                 - - ACCGTGATGA TGGCCTCGTC GGCCACCGCC GTCGCTCCGT TCCAGGGGCT CA -            #AGTCCACC    120                                                                 - - GCCAGCCTCC CCGTCGCCCG CCGGTCCTCC AGAAGCCTCG GCAACGTCAG CA -            #ACGGCGGA    180                                                                 - - AGGATCCGGT GCATG              - #                  - #                      - #   195                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 177 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - ACGTCGCTCA TGTATGTATG ATCCGTCGGT CCGTCGGTAC CGCGGGTGGC AC -             #TACTACCG     60                                                                 - - GAGCAGCCGG TGGCGGCAGC GAGGCAAGGT CCCCGAGTTC AGGTGGCGGT CG -            #GAGGGGCA    120                                                                 - - GCGGGCGGCC AGGAGGTCTT CGGAGCCGTT GCAGTCGTTG CCGCCTTCCT AG - #GCCAC           177                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - ATCACTTTCA CGGGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - ATCACGTTCA CGGCA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Ile Thr Phe Thr Gly                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - CCTTGGCAGC CATCACGTTC ACGGGAAGTA TTGTC       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - ATCTGGCAGC AGAAAAACAA GTAGTTGAGA ACTAAGAAGA AGAAA   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - CATCGAGACA AGCACGGTCA ACTTC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - AAGTCCCTGG AGGCACAGGG CTTCAAGA         - #                  - #                 28                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - GCTTACCTAC TAATTGTTCT TGG           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - CAGGGTACAT ATTTGCCTTG GG           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - AACCCTGAAT GGAAGTGC             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - ACGGACAGAT GCAGATTGG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Pro Arg Gly Ser Thr                                                      1               5                                                            __________________________________________________________________________

What is claimed is:
 1. A fertile transgenic Zea mays plant containing anisolated heterologous chimeric DNA construct encoding EPSP synthaseoperably linked to an actin promoter wherein said heterologous chimericDNA construct is expressed so that the plant exhibits tolerance orresistance to normally toxic levels of glyphosate, wherein saidtolerance or resistance is not present in a Zea mays plant notcontaining said heterologous chimeric DNA construct, and wherein saidheterologous chimeric DNA construct is transmitted through a completenormal sexual cycle of the transgenic plant to the progeny.
 2. A seedproduced by the transgenic plant of claim 1 which comprises saidheterologous chimeric DNA construct.
 3. A progeny transgenic Zea maysplant derived from the transgenic plant of claim 1 wherein said progenyplant expresses said heterologous chimeric DNA construct so that theprogeny plant exhibits said glyphosate tolerance.
 4. A seed derived fromthe progeny plant of claim 3 wherein said seed comprises saidheterologous chimeric DNA construct.
 5. The Zea mays plant of claim 1wherein the promoter is the rice actin promoter.